Antigen Binding to Secretory Immunoglobulin A Results in Decreased Sensitivity to Intestinal Proteases and Increased Binding to Cellular Fc Receptors*

In intestinal secretions, secretory IgA (SIgA) plays an important sentinel and protective role in the recognition and clearance of enteric pathogens. In addition to serving as a first line of defense, SIgA and SIgA·antigen immune complexes are selectively transported across Peyer's patches to underlying dendritic cells in the mucosa-associated lymphoid tissue, contributing to immune surveillance and immunomodulation. To explain the unexpected transport of immune complexes in face of the large excess of free SIgA in secretions, we postulated that SIgA experiences structural modifications upon antigen binding. To address this issue, we associated specific polymeric IgA and SIgA with antigens of various sizes and complexity (protein toxin, virus, bacterium). Compared with free antibody, we found modified sensitivity of the three antigens assayed after exposure to proteases from intestinal washes. Antigen binding further impacted on the immunoreactivity toward polyclonal antisera specific for the heavy and light chains of the antibody, as a function of the antigen size. These conformational changes promoted binding of the SIgA-based immune complex compared with the free antibody to cellular receptors (FcαRI and polymeric immunoglobulin receptor) expressed on the surface of premyelocytic and epithelial cell lines. These data reveal that antigen recognition by SIgA triggers structural changes that confer to the antibody enhanced receptor binding properties. This identifies immune complexes as particular structural entities integrating the presence of bound antigens and adds to the known function of immune exclusion and mucus anchoring by SIgA.

secretions, SIgA binds antigen (Ag), thus preventing its adhesion to the luminal epithelial surface and facilitating its elimination by peristalsis or mucociliary movements (2), a phenomenon called immune exclusion (3).
SIgA consists of two or four monomeric IgA linked through a joining (J) chain in association with secretory component (SC) (4). SC is derived from the polymeric immunoglobulin receptor (pIgR) that ensures selective transport of polymeric IgA (pIgA) across the epithelium to the luminal environment (5). SIgA exhibits a remarkable stability in the harsh environment of the gastrointestinal tract (6). Another feature of SIgA is its capacity to adhere selectively to microfold (M) cells in Peyer's patches (7); these latter are subsequently able to transport SIgA Ab across the epithelium and to bring them in contact with dendritic cells in the underlying mucosa-associated lymphoid tissue (8). In the form of SIgA⅐Ag immune complexes (IC), this translates into the onset of mucosal and systemic responses associated with production of anti-inflammatory cytokines and limited activation of Ag-presenting cells (9).
In face of the large excess of SIgA in the intestinal lumen, intrinsic passage of the Ab or IC remains limited. In this context, we previously postulated that upon binding of the Ag, SIgA experiences conformational changes that result in increased binding to the so far unidentified IgA receptor on the apical surface of M cells (7). Conformational differences in pIgA-or SIgA-based IC compared with the corresponding free Ab may also explain differential mucus-binding properties and immune exclusion (10). To address the question of Ag-mediated structural changes in pIgA and SIgA, we focused our analysis on biochemical approaches comparing pIgA and SIgA either as free Ab molecules or in association with three antigenic structures of increased complexity, namely a protein (Clostridium difficile toxin A), a virus (rotavirus), and a bacterium (Shigella flexneri).
In this study, we found that Ag-driven conformational changes can be evidenced by examining the differential sensitivity to intestinal proteases and immunoreactivity of IC compared with free Ab. Increased selective binding to known cellular receptors (Fc␣RI, pIgR) further revealed major changes in IC compared with free Ab. The data support the notion that binding to Ag of various sizes affects the structure of pIgA and SIgA molecules in such a manner that it makes Ag-complexed Ab molecules a better substrate for receptors that relay the function of the Ab.
Mouse secretory component (mSC) was recovered from a stable cell line (clone 2H2) by affinity chromatography on a 2-ml M2-agarose column (14). The source of human SC (hSC) was published previously (10). C. difficile toxin A was purchased from Calbiochem.
In Vitro Reassociation of pIgA and hSC or mSC-SIgA molecules were obtained by combining in vitro 10 g of mouse pIgA molecules with 2 g of hSC or mSC, respectively. Reassociation was performed in PBS for 30 min at room temperature as described previously (6). Integrity and proper assembly of the molecule were examined by SDS-PAGE under nonreducing conditions and immunodetection using antisera specific for the ␣ chain, the J chain, and SC (14,15).
Preparation of Rotavirus-The bovine rotavirus (RF strain) was propagated in monkey kidney MA104 cells in presence of trypsin as described previously (18). Virus titer present in the lysed cell supernatants was determined by plaque assay and expressed as plaque-forming units/ml. The virus was purified on a cesium chloride gradient as described before (19) and conserved at 4°C until further use. For biochemical analyses, purified rotavirus solutions were desalted immediately before use by gel filtration on Sephadex G-25 coarse beads (Amersham Biosciences), and the concentration was calculated by spectrophotometry (Ultrospec 300; Pharmacia Biotech) using the following correlation: 5.3 A 260 corresponds to 1 mg/ml virus.
Formation of IC-IC were formed as follows. 100 ng of C. difficile toxin A was mixed with 50 ng of either pIgAPCG-4 or SIgAPCG-4 permitting neutralization in in vitro assays (11). 60 ng of purified rotavirus was incubated with 100 ng of either pIgA7D9 or SIgA7D9 in PBS for 2 h at room temperature, corresponding to ϳ160 Ab molecules/virus (19). 10 8 S. flexneri were incubated with 120 ng of either pIgAC5 or SIgAC5 in PBS for 25 min on ice, corresponding to ϳ2000 Ab molecules/bacterium (20). These conditions guarantee that no free pIgA/SIgA are found in the IC preparations.
Digestion of Ab and IC with Mouse Intestinal Washes-A laparotomy was performed on BALB/c mice (4 -6 weeks old) purchased from Harlan (Den Horst, The Netherlands). The gut was cut, and the intestinal lumen was washed with 300 l of PBS. Final aspiration resulted in the recovery of ϳ200 l of intestinal wash, which was immediately aliquoted and frozen in liquid nitrogen (6). For in vitro digestion, 120 ng of purified pIgA or SIgA (pIgAPCG-4, SIgAPCG-4, pIgAC5, SIgAC5, pIgA7D9, SIgA7D9) or IC was mixed or not with 1 or 2 l of intestinal washes in a final volume of 20 l of PBS and incubated at 37°C for either 3 or 21 h. Reactions were immediately stopped by the addition of 2 l of Complete TM protease inhibitor mixture (Roche Applied Science).
Enzyme-linked Immunosorbent Assay (ELISA)-96-well plates (Nunc-immuno Plate Maxisorp surface; Nalge Nunc International, Roskilde, Denmark) were coated with a range of 20 -40 ng of either Ab, IC, or Ag in a final volume of 100 l of coating buffer (PBS or NaHCO 3 (pH 9.6)) for 2 h at room temperature. After washing three times with PBS-T, wells were blocked with PBS-T containing 1% of bovine serum albumin (Fluka, Buchs, Switzerland) for 1 h at room temperature. Detection of human or mouse ␣and chains was carried out for 1 h at room temperature with 100 l/well of the same primary/ secondary Ab/antisera in PBS-T and 1% bovine serum albumin used in Western blot assays. After final washes, revelation was performed with a 0.1 M citrate sodium solution (pH 5.0) containing 1 mg/ml o-phenylenediamine (Sigma) and 0.01% H 2 O 2 . The reaction was stopped by the addition of 2 M sulfuric acid, and the absorbance was measured at 492 nm with 620 nm as reference.
Cells-The human promyelocytic cell line U937 was obtained from the American Type Culture Collection. The cells were plated in 75-cm 2 plastic flasks containing RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 g/ml streptomycin, and 100 units/ml penicillin, at 37°C in a humidified 5% CO 2 /air incubator. The cells were subcultured every third day, at a density of 3 ϫ 10 5 cells/ ml. 18 h prior to binding experiments, cells were stimulated with 10 Ϫ8 M phorbol myristate acetate because this was shown to increase surface expression of Fc␣RI (22). Transfected Madin-Darby canine kidney (MDCK) cells overexpressing the human pIgR were cultured to 90% confluence on 6-well plastic culture dishes as described previously (23).
Flow Cytometry-U937 or MDCK cells were washed twice with PBS. 2 ϫ 10 5 cells in 100 l of PBS containing 1% fetal calf serum (PBS-S) were preincubated with 50 g/ml human or mouse IgG for 30 min at 4°C to mask Fc␥Rs. After washing with cold PBS, cells were incubated for 1 h at 4°C with 100 l of PBS-S containing 2 g of fluorescein-labeled pIgA or SIgA or the same amount of IgA in the form of IC thereof. Unbound Ab or complexes were washed three times with ice-cold PBS. Cells were gently detached with a rubber policeman, allowing for viability above 90%, as determined by trypan blue staining. For binding inhibition studies, U937 cells were preincubated with 1 g of anti-Fc␣RI mAb MIP8a (AbD Serotec, Düsseldorf, Germany), and MDCK cells with 10 l of rabbit antiserum to hSC prior to the addition of fluorescent IgA or IC. Cell-associated fluorescence intensity was evaluated by flow cytometry (FAC-Scan flow cytometer; Becton-Dickinson).
Measurement of Binding Affinity-U937 and MDCK cell-associated radioactivity was determined. Moles of bound pIgAPCG-4, SIgAPCG-4, or IC formed with the toxin A were calculated, and equilibrium dissociation constants (K d ) and number of binding sites per cell were determined by Scatchard analysis (24).
Statistical Analysis-All statistical analyses were performed using GraphPad Prism version 5. For all data, an unpaired Student' t test was applied, and the limit of significance was set at p ϭ 0.05.

RESULTS
Proper Assembly of pIgA and SIgA for Biochemical and Binding Studies-To examine the consequences of the binding of Ag of various nature (protein, virus, bacterium) on IgA structure, we generated cognate monoclonal Ab in the two molecular forms relevant for mucosal immunity, i.e. pIgA and SIgA. Purified pIgA (IgAPCG-4, IgA7D9, and IgAC5) was analyzed by immunodetection following separation on polyacrylamide gel under nonreducing conditions (Fig. 1). Proper assembly and integrity of the molecules were checked using antisera specific for the ␣ chain (lanes 1, 4, and 7) and the J chain (lanes 2, 5, and 8), which is essential for the association of the pIgA with SC. A combination of equimolar amounts of purified pIgA and hSC (IgAPCG-4) or mSC (IgA7D9 and IgAC5) for 1 h at room temperature resulted in the formation of covalent heterodimer complexes, as indicated by the detection of shifted SC to the position of pIgA (lanes 3, 6, and 9) when the protein was analyzed under nonreducing conditions. As reported previously, the percentage of covalence varied from one pIgA to another (11,25) as reflected by the appearance of free SC migrating as a band of 80 kDa. When loaded onto a molecular sieving column run in PBS, co-elution of SC and pIgA confirmed that the two proteins were indeed associated in the form of covalent and noncovalent SIgA complexes (15) (data not shown).
Impact of a Protein Ligand on IgA Structure-We have reported that binding of SC to pIgA protects the Ab from in  9) were revealed using antisera specific for mouse ␣ chain, J chain, and SC, respectively. The molecular mass (kDa) of immunoreactive species is shown alongside the lanes. In lanes 3, 6, and 9, the band at 80 kDa represents free SC not covalently bound to pIgA. JANUARY 8, 2010 • VOLUME 285 • NUMBER 2 vitro degradation by intestinal washes mimicking the proteaserich environment found in vivo (6). We used the same technical approach to evaluate the impact of C. difficile toxin A binding on the sensitivity of pIgAPCG-4 and SIgAPCG-4, as reflected by the appearance of ␣ and chain degradation products assayed by immunodetection (Fig. 2, A and B). In time course experiments, the susceptibility of SIgAPCG-4 to intestinal washes was reduced compared with pIgAPCG-4, with integral ␣ chain (62 kDa band) recovered after 21 h of incubation in addition to degradation products of 40 and 36 kDa (Fd fragment). Upon association with the toxin A Ag, the pattern of digestion of the two molecular forms of the Ab was the same, with undegraded ␣ chain detected at the 21-h end point. The C-terminal cleavage of the ␣ chain, yielding the 36-and 40-kDa fragments, was shown to occur upon incubation with intestinal washes (6), some distance away from the Fab domains. This suggests that the susceptibility of the protein is reduced through the preferential action of conformational changes rather than masking of sensitive sites. In contrast, the integrity of the chain was not affected under any experimental conditions, arguing intramolecular and toxin A-mediated steric hindrance ensuring protection against the action of proteases (Fig. 2B).

Structural Changes in SIgA-based Immune Complexes
Changes in conformation and topography in pIgA and SIgA molecules have been assessed by analyzing the immunoreactivity of the constituting polypeptides in the Ab (26). Hence, we compared the reactivity of free and toxin A-bound pIgAPCG-4 and SIgAPCG-4 with antisera specific for either the ␣ or the chains by ELISA (Fig. 2C). We chose to use polyclonal antisera to permit to map the overall effect of toxin A binding on the availability of linear and conformational epitopes. For both chains, a weak (16 -18%) yet highly reproducible reduction in reactivity was detected upon toxin A binding. The effect was slightly reinforced in the presence of SC in the Ab molecule (23-25% reduction), possibly accounted for by the capacity of SC to bind with toxin A (19) or masking of epitopes on the ␣ chain. Together, this reflects changes directly associated with the impact of the bound Ag. Furthermore, the assay is complementary to that based on exposure to protease because it also implicates the chain in the process.
Impact of a Whole Virus Ligand on IgA Structure-We next investigated whether the variability in protease sensitivity and immunoreactivity of pIgA/SIgA we observed after association with a protein Ag would similarly occur in the context of Ag of much bigger sizes and thus epitopic complexity. In comparison with the "naked" Ab that remained degradable into low molecular mass ␣ chain products, incubation of rotavirus with VP6 (surface-exposed virus protein 6)-specific pIgA7D9 or SIgA7D9 led to the demonstration that the Ab in either molecular form was almost fully protected against the action of proteases in intestinal washes (Fig. 3A). However, for this particular IgA, the addition of SC did not offer a better protection against proteases; this might be due to the low percentage of covalent interaction between SC and the ␣ chain, or alternatively, to the intrinsic stability of IgA7D9 in the assay (Fig. 1). Together, these features may partially mask the impact of Ag binding in this assay. In support of a less reduced protective effect of SC associated with IgA7D9, reduction of the chain signal was observed after 21 h of incubation both in the absence and in the presence of SC. Tight interaction with globular rotavirus in a Fab-dependent manner maintained a stable level of chain comparable between lanes (Fig. 3B). A clearer picture could be drawn from the immunoreactivity assay. Compared with IgAPCG-4, more marked changes in the capacity to be recognized by a chain-specific antiserum were measured for pIgA7D9 (30% reduction) and SIgA7D9 (34%) (Fig. 3C). The decrease in ␣ chain reactivity can be explained by reduced access to the IgA-associated epitopes as well as by conformational effects limiting productive interactions with the antiserum, both resulting from coating of the virus particle by the Ab. In pIgA7D9, chain in close proximity with the virus Ag displayed a reduction in reactivity (18%), favoring the masking hypothesis over distant structural changes in this case. In SIgA7D9, the drop in immunoreactivity reached 22%, in support of the spatial distribution of SC wrapping the constant domains of the protein (27).
Impact of a Whole Bacterium Ligand on IgA Structure-Comparison of free pIgA/SIgA and the corresponding IC made of a protein or a viral Ag indicates that close interaction between partners on one hand and induced subtle modifications in the Ab structure on the other hand are differently affected as a function of the Ab⅐Ag couple. We further challenged this notion by extending our analysis to the effect of associating S. flexneri to IgAC5 specific for the bacterial surface lipopolysaccharide. Upon Ag binding, both molecular forms of the IgA Ab behaved the same, with a mixture of undigested ␣ chain and 36-and 40-kDa degradation products detected at the two time points (Fig. 4A). This contrasted with the pattern obtained in the absence of S. flexneri, where unexpectedly, it appeared that pronounced degradation of the Ab took place at 21 h irrespective of bound SC (Fig. 4A). Although some crossreactivity between the anti-␣ chain Ab and S. flexneri proteins could be observed, its contribution to the specific signal could be neglected in terms of interpretation of the results. The sta-bility of the chain was demonstrated under all experimental conditions (Fig. 4B).
The immunoreactivity of the ␣ chain was about the same between pIgA and SIgA (Fig. 4C), suggesting a loose, although covalent association of SC, consistent with the limited protection observed in Fig. 4A. In contrast, a 26% reduction of chain reactivity occurred upon association with SC; this might result from a particular folding of the Fab domains in SIgAC5 because the first molecular model for SIgA shows surface exposure of two of four chains (27). The combination of the bacterium with the Ab led to a marked drop in ␣ chain immunoreactivity for both the polymeric (91%) and secretory (92%) forms of IgAC5. A slightly less pronounced reduction for chain was measured for pIgAC5 (81%) and SIgAC5 (86%). In complexes with S. flexneri, these changes could be caused by enhanced epitope density recognized by the IgAC5 Ab or, alternatively, by masking of proteolytic sites in the ␣ and chain.
Binding of Free IgA and IC to Cellular Receptors-Results obtained by exposure to intestinal washes and immunoreactivity argue in favor of multiple consequences on the structure of the pIgA/SIgA Ab molecules following interaction with Ag of various sizes and nature. At the biochemical level, these combined approaches led us to conclude on the dichotomy between structural changes and steric hindrance. To gain further insight into the impact of Ag binding on pIgA/SIgA molecules, the interaction with two known receptors of the Ab was evaluated using cell lines expressing Fc␣RI or pIgR. We focused our analyses on toxin A-pIgA/SIgA IC because surface distribution of the fluorescent Ab on the rotavirus and S. flexneri Ag would bias the assessment of the mere contact between the receptor and the Ab ligand. For the duration of the experiment, we initially checked that toxin A assayed alone was not toxic for the  Fig. 2, A and B, is shown, with the exception that pIgA7D9 and SIgA7D9, complexed or not with rotavirus, is assayed by immunodetection. The background signal due to cross-reactivity with viral antigens is shown on the two last lanes. One representative experiment of three is depicted. C, change in ␣ chain and chain reactivity of pIgA7D9 or SIgA7D9 molecules in the absence or presence of rotavirus, respectively. Data are expressed compared with the absorbance mean values obtained for pIgA7D9 fixed arbitrarily at 100. Bars represent mean values Ϯ S.D. (error bars) (n ϭ 4). Significant statistical differences are indicated above the bars.
cells (data not shown). Fluorescein-labeled chimeric pIgAPCG-4/SIgAPCG-4 carrying human constant domains was examined by flow cytometry for its capacity to recognize Fc␣RI present on the surface of the monocytic cell line U937 (Fig. 5A). Consistent with previous data, both molecular forms of the Ab bound the cell line (Ͼ95% fluorescent cells), reaching uniform fluorescence levels well above background measured with medium alone. Binding, as measured by an increase in mean fluorescence intensity, of either pIgAPCG-4 or SIgAPCG-4 Ab was almost doubled in the presence of bound Ag (Fig. 5A). Association of free or antigen-bound fluorescent Ab molecules to U937 cells was strongly reduced when Fc␣RI was blocked by preincubation with the specific mAb MIP8a (Fig. 5A). Together, this indicates that binding of the protein Ag somehow alters the structure of either the polymeric or secretory form of the Ab in such a way that it enhances the specific association with U937 cells expressing Fc␣RI.
The same set of experiments was conducted using epithelial MDCK cells overexpressing human pIgR (Fig. 5B). As expected, in the absence of toxin A protein, pIgAPCG-4 bound efficiently to MDCK cells, whereas SIgA was unable to do so as a consequence of the SC moiety present on the Ab (Fig. 5B). Similar to the picture with Fc␣RI, antigen association resulted in a 2-fold increment in pIgAPCG-4 Ab binding to cells expressing human pIgR. SIgA-based IC did not bind, indicating that the toxin A Ag did not perturb the arrangement of SC. Specificity of association between pIgR and pIgAPCG-4 was further confirmed in blocking experiments carried out in the presence of anti-human SC antiserum (Fig. 5B). Both cellular receptors exhibit improved binding properties for IC compared with the Ab alone. This indirectly suggests that accessibility is not limiting, and differences may involve relative affinity. Scatchard analyses revealed binding sites of higher affinity for IC compared with Ab alone (Table 1). Because the same cell lines with the same absolute number of receptors were used, this implies that more effective binding of IC occurred; structural changes triggered by the binding of the antigen are likely to be the cause of the differences observed.

DISCUSSION
The data reported herein demonstrate that, upon Ag binding, pIgA and SIgA experience structural changes reflected in the differential sensitivity to intestinal proteases and immunoreactivity toward ␣ and chain-specific antisera. Furthermore, Ag binding increased the capacity of the Ab molecule to interact with two well documented IgA cellular receptors, namely Fc␣RI and pIgR. Thus, the conformational changes characterized by epitope recognition, protease sensitivity, and binding to cellular receptor have physiologic consequences particularly relevant to mucosal immunology.
The nature and size of the Ag did not seem to be crucial parameters for these structural changes to occur because we found measurable effects when using a protein, a virus, or a bacterium in association with their cognate pIgA/SIgA Ab. The formation of IC of higher size range due to an increasing number of anchoring sites present on the Ag did indeed reduce enzymatic degradation of the ␣ chain, a phenomenon accompanied by a decrease in epitope recognition. In addition, structural "freezing" of the Ab molecule upon interaction with the Ag might as well explain the differences found with respect to sensitivity to proteases or diminished recognition by antisera. Because interaction with cellular receptors was improved, we believe that conformational changes mediated by Ag binding are principally responsible for the observed effects, with steric hindrance playing a limited role. Moreover, this is consistent with the more artificial situation where aggregated pIgA exhib- ited a better binding to target cells (28), possibly because of reduced dissociation of polymers as seen in our data and Ref. 29. In the context of IC, Ag recognition brings together Ab molecules and thus increases productive interactions with cellular receptors.
We reported in a previous study that IgA-based IC are taken up by intestinal M cells in Peyer's patches (20). Although the receptor(s) involved is (are) in need of identification, the phenomenon is specific for the IgA isotype (mouse IgA and human IgA2 only) and requires both the Fc␣1 and Fc␣2 domains of the Ab (7). In face of the large excess of free SIgA in mucosal secretions, one would have expected that sampling by M cells is an unlikely event. We speculated that preferential uptake of SIgA⅐Ag complexes compared with free SIgA might be due to conformational change(s) that favor(s) binding to the M cell receptor. In support of our working hypothesis, we found that binding to Ag resulted in decreased sensitivity of the Ab to the action of proteases in the intestinal washes, indicating that domains in the Fc region were most likely in a more closed conformation or at least presented less accessible cleavage sites for intestinal enzymes. The effect was limited to the C-terminal portion of the Ab molecule because the light chain buried in the Fab was not, or only slightly, affected by the incubation with intestinal washes. An immunoreactivity study led to a decline in ␣ chain and chain recognition upon Ag binding, and the effect was even more pronounced for SIgA, as expected from the wrapping of SC around the Fc domains of pIgA (27).
Increased interaction with pIgR and Fc␣RI of IC further indicates that important structural modifications occurred, which might also have relevance for the sampling of Ag by M cells. If we assume a multivalent nature for the interaction between pIgA/SIgA-based IC and the M cell receptor(s), this would lead to a slow off-rate, allowing stable binding and subsequent internalization, even in the presence of the high free SIgA concentrations found in exocrine secretions. The same mechanism has been postulated to explain Fc␣RI-mediated phagocytosis by monocytes in serum containing large excess of circulating IgA (30).
We observed identical association of free pIgA and SIgA with Fc␣RI on the surface of U937 cells. This confirms previous data

Structural Changes in SIgA-based Immune Complexes
showing binding of either form of the Ab, yet in a less quantitative manner (31). Although residues involved in the binding to Fc␣RI seem to also interact with SC (32)(33)(34), the expression of the co-receptor Mac-1 (CD11b/CD18, complement receptor 3) (35) on the surface of U937 stimulated with phorbol myristate acetate is sufficient to guarantee recognition of SIgA as well. In the form of IC, both pIgA and SIgA bound more efficiently to U937 cells, indicating that conformational changes most likely relayed by the Fab domains promoted interaction with Fc␣RI. This is consistent with structural variability mapped in Fc␣RI upon interaction with the recombinant Fc␣ fragment consisting of domains C␣2 and C␣3 (30). The importance of the C␣2/C␣3 junction as sites of functional diversity is indirectly reflected by the fact that it appears to be a hot spot for targeting of molecules produced by pathogens; such a mechanism of subverting immunity has been documented for Staphylococcus aureus SSL7 (36), group A streptococcus Sir22 (37), and Arp4 (38), as well as group B streptococcus b Ag (39). We also found enhanced binding to pIgR of pIgA-based immune complexes compared with free pIgA. This may be rationalized by the need to transport pIgA⅐Ag complexes efficiently from the lamina propria to the lumen to preserve mucosal integrity (5).
In contrast to our results, Kaetzel et al. (24) found similar K d values for binding of pIgA⅐IC and free pIgA to rabbit pIgR. This could be due to species differences between human and rabbit pIgA-pIgR interactions: rabbit pIgA has a very high affinity for its cognate pIgR, and it may therefore not be necessary to enhance binding of IC to expedite export of foreign Ag. Possible technical explanations for these differences range from the Ag:Ab ratio used, the nature and size of the Ag, the Ab coating by the Ag, and from the number and nature of pIgR. Together, these data indicate that subtle changes in the overall conformation of pIgA and SIgA take place upon Ag binding, resulting in modified protease sensitivity, epitope exposure, and interaction with cellular receptors. This strongly suggests that similar events can explain the differential binding of Agloaded SIgA compared with "free" SIgA to the still to be identified receptor at the surface of M cells in intestinal Peyer's patches. More generally, this raises the concept that when the Ab is complexed to the Ag, improved interactions with cellular receptors are established, which might eventually impact on the biological function of the target cells.