Conserved regions of protein disulfide isomerase are targeted by natural IgA antibodies in humans

Bob Meek1, Jaap Willem Back2, Vincent N. A. Klaren1, Dave Speijer3 and Ron Peek1

1 Department of Molecular Immunology, The Netherlands Ophthalmic Research Institute, Meibergdreef 47, 1105 BA Amsterdam, The Netherlands 2 Swammerdam Institute for Life Sciences, Mass Spectrometry Group, and 3 Department of Biochemistry, University of Amsterdam, 1105A2 Amsterdam, The Netherlands

Correspondence to: B. Meek; E-mail: b.meek{at}ioi.knaw.nl
Transmitting editor: T. Honjo


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Secretory IgA (sIgA) antibodies in human tears and milk were found to recognize protein disulfide isomerase (PDI) on a Toxoplasma gondii lysate immunoblot (IB). These antibodies were already detectable in tears of infants. To determine the epitope containing-regions on PDI, we generated truncated versions of recombinant PDI that differ by 8–10 amino acids in length. By IB, it was found that the sIgA epitopes were confined to conserved regions of PDI, including the functionally essential thioredoxin-like domain. This suggested the capacity of sIgA to react with PDI of other species, which was confirmed by recognition of human PDI by IgA in tears. In contrast, anti-T. gondii PDI antibodies generated by immunization were not able to cross-react. Binding to the thioredoxin-like domain on IB could be gradually abrogated by incubation with peptide constituting the same domain. By consecutive investigation of the function of the protein targeted by sIgA, the presence of antibody in relation to age and analysis of the epitope constituting regions on PDI we demonstrate that sIgA directed against PDI are self-reactive natural antibodies. Furthermore, analysis of antibody epitopes on an antigen is a useful method to distinguish conventional, affinity-matured antibodies from natural antibodies. The presence at early age and continuity of anti-PDI sIgA in relation to age suggests the existence of B cells secreting germline-encoded antibodies in human mucosa outside of the gut. Overall, the PDI-specific antibodies are clearly part of the natural antibody repertoire, suggesting an active role for these antibodies in the innate defense against pathogens.

Keywords: antibody, antigen, cross-reactivity, epitope, human, lacrimal gland, parasitic protozoan, peptide, repertoire development


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The humoral component of the mucosal immune system (MIS) mainly consists of secretory IgA (sIgA) and IgM antibodies. The most important functions of these antibodies are ‘immune exclusion’ and ‘immune elimination’, achieved by binding to surface antigens on potential pathogens. ‘Immune exclusion’ refers to prevention of adherence to epithelial cells and subsequent infection, while ‘immune elimination’ involves pathogen clearance in the lamina propria and neutralization of intracellular viruses (13). Both conventional (monospecific) and natural (polyspecific) antibodies are important for optimal protection against pathogens (2,4).

Conventional antibodies are produced by B2 cells and are affinity matured upon exposure to specific antigens at inductive sites of the MIS, such as the Peyer’s patches in the ileum, the mesenteric lymph nodes (MLN) and the tonsils. Once induced, mature B cells migrate to effector sites that can be located distant from the inductive site (57). Whether specific IgA can be detected at mucosal surfaces apart from those close to an inductive site depends on immunogenicity and quantity of the antigen(s) presented at these site(s) (5,7). Natural antibodies can be found in any mucosal secretion in man and mice. Characteristically, natural antibodies target phylogenetically conserved structures of commensals, pathogens and autoantigens (810). Most murine B cells secreting natural antibodies at mucosal sites express their antibodies in or near the germline configuration (11). In humans, there does not seem to be a well-defined population of B cells secreting IgA antibodies in the germline configuration (1215), although not all mucosal sites have been studied. Nevertheless, natural polyreactive IgA have been demonstrated in human mucosal secretions (10,16). General features of both natural and conventional antibodies recognizing proteins are summarized in Table 1.


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Table 1.
 
In a recent study we found that IgA produced by the human lacrimal gland predominantly recognize 49-, 70- and 74-kDa antigens of Toxoplasma gondii (17). The 49-kDa antigen was identified as protein disulfide isomerase (PDI) (18), an essential enzyme that functions in the endoplasmic reticulum of eukaryotic cells (19). T. gondii is prevalent in northwestern Europe (20), where the previous studies were conducted, resulting in a gradual increase in prevalence of anti-toxoplasma serum IgG with age (20,21). Toxoplasmosis is usually self-limiting and benign in immunocompetent individuals, but may cause serious complications in immunocompromised patients and neonates (20). Penetration of the gut epithelial barrier by T. gondii is required to initiate systemic infection, and is usually accompanied by a vigorous common mucosal sIgA response in humans and mice (22,23). This suggests that the anti-toxoplasma IgA in tears are the result of occasional encounters with T. gondii and implicates that they are conventional antibodies. On the other hand, the anti-toxoplasma IgA staining pattern on immunoblot remained remarkably stable in time (17) and PDI is a highly conserved protein (19), implying a natural origin of these antibodies. Since PDI has both species-specific as well as highly conserved regions, we hypothesize that conventional antibodies are preferentially directed against T. gondii-specific, non-‘self’ regions, while natural antibodies will primarily recognize conserved regions of T. gondii PDI, presumably being molded by ‘self’ antigens.

The relation between age and presence of these antibodies in tears was determined to see whether they conformed to the characteristics of conventional or natural antibodies as summarized in Table 1. Next, we tested our hypothesis by identifying the protein regions involved in antibody recognition and determined whether there was cross-reactivity with PDI from other species. Our findings strongly support an innate origin for these antibodies. To our knowledge, this is the first report of a detailed analysis of a natural antibody response in man.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Volunteers and sample collection
Tear fluid was collected from eyes of healthy volunteers with glass capillaries (Assistent; Karl Hecht, Sondheim, Germany) (24). Spontaneously produced tears were collected from infants by their parents. Tears were stored at –20°C, until use. Paired tear fluid and milk samples were obtained from three women at different stages of lactation. Tears of adults and infants were diluted 200 and 150 times respectively, except when stated otherwise. Human milk was diluted 25 times. This study was conducted in accordance with the Declaration of Helsinki.

Parasites
T. gondii tachyzoites of the RH strain were propagated in vitro under normal cell culture conditions in RK13 cells in RPMI medium supplemented with 3% FCS. Plasmodium falciparum schizonts and gametocytes were provided by H. Eling (Academic Hospital Nijmegen, Nijmegen, The Netherlands).

Immunofluorescence (IF)
IF was performed as described earlier (25), except that parasites were not fixated. Briefly, 105 filter-purified parasites were double-labeled by incubation with tears and a mouse mAb against surface antigen (SAG) 1 (HyTest, Turku, Finland), diluted 500 times, in PBS containing 2% BSA, at 4 °C for 30 min. Following washes, parasites were incubated with anti-human IgA conjugated to FITC, diluted 50 times, and anti-mouse Fab conjugated to indocarbocyanine (Cy3), diluted 750 times, for 30 min. Following washing and drying, labeled parasites were embedded in Vectashield (Vector, Burlingame, CA) and visualized using a laser scanning microscope (Zeiss LSM 410; Carl Zeiss Microscopie, Göttingen, Germany).

Preparation of antigens
T. gondii tachyzoites were filter purified using filters with a pore size of 3 µm supplemented with a pre-filter. P. falciparum parasites were provided as pellets and resuspended in PBS with protease inhibitors before use. Human embryonic kidney cell extract derived from cell line 293T was provided by C. Neefjes (NORI, Amsterdam, The Netherlands). Parasites were processed as described (17). The extracts of P. falciparum and the human cell line were concentrated 8–10 times to allow detection of PDI.

SDS–PAGE, Western blotting and detection of peroxidase-conjugated antibodies
Procedures were performed as described earlier (17,26). ß-Mercaptoethanol was added to a concentration of 5% prior to electrophoresis. Following this, proteins were transferred to PVDF membranes. Membranes were blocked with Tris-buffered saline (TBS: 50 mM Tris pH 10, 150 mM NaCl), containing 0.5% Tween 20 and 2% non-fat milk powder. All samples and conjugates were diluted in TBS containing 0.5% Tween 20 and 0.03% non-fat milk powder (TBS-T). A peroxidase-conjugated polyclonal antibody preparation specific for human secretory component was diluted 4000 times. Bound antibodies were visualized by chemiluminescence, according to the manufacturer’s description (ECL; Amersham Biosciences, Little Chalfont, UK) and exposed to X-ray film.

Primers and peptides
For primers, see Table 2. Synthetic peptide FYAPWCGHCK-COOH was manufactured commercially (Pepscan Systems, Lelystad, The Netherlands). PCR products were ligated into the pGEM-T Easy cloning vector as described by the manufacturer (Promega, Madison, WI) or digested and ligated into expression vector pRP261, a derivative of vector pGEX-3X (Amersham Biosciences).


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Table 2. Primers used for the production of truncated PDI
 
Construction of the PDI expression vectors (Fig. 2)
Construction of vectors expressing full length and PDI {Delta}2 has been described previously (18). The PDI {Delta}1 mutant was obtained after EcoRI digestion of full-length PDI in pRP261 and subsequent ligation (see Fig. 2). To determine the location of the antibody-binding regions in PDI {Delta}2, the fragment encoding PDI {Delta}2 was split in two parts, named PDI {Delta}3 and PDI {Delta}4. PDI {Delta}3 was generated by digestion of pRP261-{Delta}2 with SalI and NcoI, followed by treatment with 1 U S1 nuclease in 100 mM NaCl for 30 min at 30°C and subsequent ligation. The vector expressing PDI {Delta}4 was generated by digestion with SalI, Klenow filling and further digestion with SmaI.

For fine mapping of the {Delta}4 region, truncated versions of PDI {Delta}4 were constructed by cloning NcoI- and EcoRI-digested PCR products, obtained with PDI 12 as forward primer and PDI 13–21 as reverse primers into pRP261 (see above).

Expression, purification and detection of recombinant proteins coupled to GST
All proteins were expressed in Escherichia coli strain BL21 according to standard procedures (27). Expression was induced by adding isopropyl-ß-D-thiogalactopyranoside (1 mM) at 30°C. GST fusion proteins were purified using glutathione Sepharose 4B beads according to the manufacturer’s instructions (Amersham Biosciences). For protein gels, beads were directly solubilized in sample buffer (26). The amount of recombinant protein used for immunoblots was normalized based on the intensity of Coomassie gel staining and staining with anti-GST antibody on blot. For the detection of GST fusion proteins, antibodies were removed using erase buffer [62.5 mM Tris, pH 6.8, 100 mM ß-mercaptoethanol and 2% (w/v) SDS] for 1 h at 70°C. Subsequently, blots were blocked for 1 h and incubated with anti-GST–peroxidase (diluted 10,000 times). Visualization by chemiluminescence was performed described above.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Anti-T. gondii IgA in tears and milk samples
Different mucosal samples were analyzed to determine whether the secretion of PDI-specific IgA antibodies was a phenomenon restricted to the lacrimal gland or a more general feature of the common MIS. The latter seems to be the case as PDI-specific IgA was detectable in both milk and tear samples of lactating women (Fig. 1, M2 and M3), although human milk had to be used at low dilutions (25 times) compared with tears (200 times).



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Fig. 1. Tear and milk antibody staining patterns on T. gondii lysate blots. To determine whether secretion of anti-PDI antibodies was correlated with age or mucosal site, tear samples of siblings D1–3 (aged 6, 9 and 11 years), infants B1 and 2 (aged 3 and 5 months), mothers M1–3, father F, and volunteers V1–7, human milk samples (Milk) and serum sample (Ser) were incubated on T. gondii lysate blots. The serum sample used was a pool of four sera obtained from patients recently infected by T. gondii. All samples were stained for anti-toxoplasma IgA, except for the ‘Ser’ and ‘SC’ lanes where samples were stained for anti-toxoplasma IgG and anti-secretory (SC) IgA antibodies respectively. The antibody specifically generated against recombinant PDI stained a 49-kDa antigen, as expected ({alpha}-PDI). Samples M1 and V7 were diluted 500 times; Ser, 1000 times.

 
In an earlier analysis of tears, we only included adults. In order to shed light on the natural or conventional origin of anti-T. gondii IgA, we analyzed tears of children for whom it was highly unlikely that they already were exposed to T. gondii [prevalence of anti-toxoplasma IgG is very low in these age groups (21)]. Anti-T. gondii IgA staining patterns from tears obtained from children and infants were compared with those of adults (Fig. 1). We readily detected T. gondii PDI-specific IgA antibodies in tears of young children (D1, D2 and D3, aged 6, 9 and 11 years) as well as in tears of infants fed exclusively on human milk (B1 and B2, 3 and 5 months of age respectively). The presence of sIgA in infant tears is not surprising as infants already produce (natural) sIgA antibodies within 3 days post partum (28).

Unexpectedly, three sisters of 6, 9 and 11 years of age displayed an almost identical IgA staining pattern of total T. gondii extract blots (Fig. 1, D1–3), with both clear differences and similarities in comparison to parental IgA staining patterns (Fig. 1, M1 and F). In addition, the tear IgA staining pattern of the infant brother (Fig. 1, B1) not only differed markedly from all his siblings, it also retained its simplicity even after long exposures; only PDI and the other common constituents of tear IgA staining patterns comprising a T. gondii 70/74-kDa antigen doublet (17) being recognized by IgA. The paucity of antigens recognized and the predominance of anti-PDI antibodies in tears was confirmed with a tear sample from an unrelated infant (Fig. 1, B2).

Finally, we confirmed that anti-PDI antibodies are of the sIgA class using a polyclonal antibody specific for secretory component (SC; Fig. 1, V7).

Expression and analysis of regions involved in epitope formation
In view of the high percentage (~80%) of volunteers with anti-T. gondii PDI IgA in their tears (17), it became important to determine whether different PDI regions were recognized by IgA in tears of different individuals. Assuming natural antibodies are not specific for T. gondii PDI, their ability to cross-react with PDI of other species would imply that natural antibodies should be mainly directed against conserved regions of PDI. To identify T. gondii PDI regions involved in IgA epitope recognition, mature PDI lacking the N-terminal signal sequence was expressed as a recombinant GST fusion protein (Fig. 2). In addition, series of PDI mutants truncated at either the N- and/or C-terminal end were constructed. Strips of Western blots with recombinant PDI were incubated with tears from individuals at dilutions resulting in recognition of PDI on a T. gondii lysate Western blot. Tear IgA from all individuals tested recognized the full-length PDI and various large fragments of PDI (PDI {Delta}1, PDI {Delta}2 and PDI {Delta}4) with the exception of the region between amino acids 402 and 471 (PDI {Delta}3, see Figs 2 and 3).



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Fig. 2. Schematic representation of native (top) and recombinant structures encoding PDI. PDI was expressed without the predicted signal sequence. Amino acid positions indicated at each N- and C-terminus refer to their position in the translated sequence of PDI, the predicted signal sequence included. GEEL is the presumed endoplasmic reticulum retention signal.

 
To further delineate the epitope(s) constituting regions within PDI {Delta}4 (amino acids 266–400) several truncated proteins were produced having their C-terminal end within this region (PDI {Delta}4, {Delta}10–12). As shown in Fig. 3 (left panels) all but one sample (F) failed to bind to PDI {Delta}10, indicating that the regions involved in IgA epitope formation were located at the C-terminal end of PDI {Delta}4, and not in the common sequences of PDI {Delta}1 and PDI {Delta}4. Importantly, the region present in PDI {Delta}4 and absent in PDI {Delta}10 (amino acids 351–400) contains the thioredoxin-like domain, a motif highly conserved in eukaryotic PDI sequences (19) (see Fig. 4).



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Fig. 3. Recognition of truncated versions of recombinant T. gondii PDI by IgA in tears. Each sample contained IgA that recognized PDI {Delta}2. Subsequently, samples were incubated with strips containing stepwise truncated (~90 amino acids) versions of PDI {Delta}2: PDI {Delta}4 and {Delta}10–{Delta}13 (see Fig. 1). All samples that recognized PDI {Delta}4 only were subjected to additional fine mapping with stepwise truncated (± 8 amino acids) versions of PDI {Delta}4: PDI {Delta}5–{Delta}9 (see Fig. 1). Strips were stained for GST after the PD-specific antibodies were removed (upper panels marked {alpha}-GST). Fine mapping demonstrated that PDI {Delta}6 and {Delta}9 contain regions essential for binding of IgA antibodies. In cases of PDI {Delta}9 recognition, PDI {Delta}8 signals were always considerably weaker than PDI {Delta}7 and {Delta}9 signals. Subject ID and dilutions of tears used correspond with Fig. 1.

 


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Fig. 4. Clustal W (1.81) multiple sequence alignment of T. gondii, P. falciparum, Cryptosporidium parvum and Homo sapiens PDI. Overall, the protein has the highest similarity to PDI from other species of the phylum Apicomplexa: P. falciparum (AJ250363) and C. parvum (U48261), with 210/444 and 171/448 identities respectively, followed by PDI homologues of Ostertagia ostertagi (AJ419174), Humicola insolens (P55059) and Onchocerca volvulus (U12440). There is considerable homology with human PDI (P55, P07237) as well: 163/453 identities. Of the five domains of PDI denoted A–B–B'–A'–C (102–60–68–104–16 amino acids), the A domains characteristically display the highest similarity with other PDI, mainly due to conservation of the thioredoxin-like domains (32,33). The alignment pattern of the A' domain is similar to the overall alignment, with T. gondii PDI (AJ306291) having most homologues with A' domains of the above-mentioned protozoan family members (56/104 and 59/104 identities respectively) and human PDI (53/105 identities). Remarkably, the A domain of T. gondii displays more homology with PDI of O. ostertagi (62/102 identities), Caenorhabditis briggsae (AJ005807, 61/102 identities), Schistosoma mansoni (S34275, 61/100 identities) and Caenorhabditis elegans (S71862, 57/102 identities) than with P. falciparum (54/101 identities). In contrast to the A regions, both B regions of T. gondii have only limited homology with B regions of PDI of P. falciparum (17/58 and 29/61 identities respectively). A and A' regions, B and B' regions, and C region of human PDI according to (48).

 
Additional truncated PDI proteins (PDI {Delta}5–{Delta}10), differing by only a few amino acids in length (Fig. 2), allowed much more precise localization of regions essential for IgA epitope formation. Two major epitopes were delimited within this region, represented in the 9-amino-acid difference between PDI {Delta}6 and {Delta}7 (amino acids 375–384) and the 8-amino-acid difference between PDI {Delta}9 and {Delta}10 (amino acids 351–359, Fig. 3). The sequence between PDI {Delta}6 and {Delta}7 contains the thioredoxin-like domain with the H -> Y amino acid substitution (18), while the sequence between PDI {Delta}9 and {Delta}10 is a more hydrophobic region with a VKVVV motif. This region is also particularly well conserved between T. gondii and various known human PDI sequences (cf. entries XM 053104.1, BC 000425.1, XM 016522.2, AL356378.17 and U75886.1, and Fig. 4). The sibling analysis depicted in Fig. 3 (D1 and D2) demonstrated that the thioredoxin-like domain is the main IgA epitope within PDI {Delta}2 for the two sisters. The newborn brother (B1) did not display IgA reactivity against this region but, similar to the mother (M1), relied on the VKVVV motif present between PDI {Delta}9 and {Delta}10 (amino acids 351–359).

These results show that healthy human subjects produce sIgA recognizing at least three different highly conserved regions on T. gondii PDI, including the thioredoxin-like domains.

IgA in tears recognizes thioredoxin-like domains, and cross-react with human and P. falciparum proteins
To confirm recognition of thioredoxin-like domains by IgA in tears, tear samples of V1 and V7 were incubated with blots of truncated PDI with (PDI {Delta}6) and without (PDI {Delta}7) a thioredoxin-like domain. Subject V1 was already shown to recognize only PDI {Delta}6 in this region and not the slightly smaller truncated versions lacking this domain (Fig. 3). Subject V7 had an anti-PDI staining pattern similar to M1 and B1 (Fig. 3), recognizing PDI {Delta}9 containing the VKVVV motif. This motif is present in both PDI {Delta}6 and {Delta}7.

Before incubation, peptide FYAPWCGHCK representing the thioredoxin-like domain was added in various concentrations. In both cases, increasing amounts of peptide resulted in a marked decrease in recognition of PDI {Delta}6, while recognition of the other PDI versions was hardly affected (Fig. 5A). This confirmed that IgA in tears from subject V1 recognized the thioredoxin-like domain and indicated that these particular antibodies form part of the repertoire of subject V7 as well. The PDI {Delta}7 recognition of subject V7 and the ‘full’ recognition of subject V1 remained intense despite the absence of a contribution by the thioredoxin-like domain. It seemed that a reduction in binding to the thioredoxin domain is compensated by an increase in binding to the VKVVV motif. Possibly, binding to these different nearby IgA epitopes in PDI is mutually exclusive.



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Fig. 5. Recognition of a thioredoxin-like domain and analysis of cross-reactivity of tear IgA antibodies. (A) Tears of subjects V1 and V7 were incubated with Western blots containing full-length PDI, and two truncated versions with (PDI {Delta}6) and without (PDI {Delta}7) the thioredoxin-like domain. Before incubation peptide FYAPWCGHCK was added in the amounts indicated. Strips were stained for GST after the PDI-specific antibodies were removed (lower panel marked {alpha}-GST). Note that the positions of the bands do not reflect the actual mol. wt. of the proteins, as small blot-strips were used in an attempt to minimize volumes required. Subject ID corresponds with Fig. 1. (B) Strips with Plasmodium schizont extract (pl), human embryonic kidney cell extract (hu), PDI {Delta}2 ({Delta}2) and T. gondii lysate (tg) were incubated with diluted tears of B1 and V1, and polyclonals specific for bovine and T. gondii PDI. The ‘tg’ strips were taken from another blot. Strips incubated with tears from subject B1 were incubated with the polyclonal specific for bovine PDI after tear antibodies were removed. Even upon long exposure, anti-bovine and T. gondii PDI polyclonals showed no cross-reactivity. Cross-reaction by IgA in tears occurred despite considerably lower anti-PDI concentrations: tears were diluted 100 times compared with 4000 and 20,000 times for bovine and T. gondii PDI respectively.

 
More importantly, recognition of the highly conserved thioredoxin-like domains by subjects V1 and V7 implies species cross-reactive capabilities. To study cross-reactivity, we compared the reactivity of tear IgA with the reactivity of two polyclonal antibody preparations directed against T. gondii PDI and bovine PDI, the latter known to cross-react with human PDI. The anti-T. gondii PDI polyclonal generated in rabbit revealed bands at expected heights in our preparations of tachyzoites and recombinant T. gondii PDI, but not in the human cell line extract or the P. falciparum extract (Fig. 5B). The polyclonal against bovine PDI reacted with a 55-kDa antigen in the human cell extract, but not with antigen in the other preparations. In marked contrast, the IgA staining patterns of infant B1 and volunteer V1 on blots containing human or Plasmodium extracts were remarkably similar in their complexity (Fig. 5B). The patterns had bands at the expected heights for human, T. gondii and P. falciparum PDI (Fig. 5B). A third sample, V7, did not show such a complex staining pattern on strips with human or plasmodium extract, but also stained bands at expected heights for the various PDI (results not shown).

T. gondii PDI is a surface antigen
To confirm our previous finding that anti-toxoplasma IgA are capable of binding to the parasite surface and thus be functional in defense against T. gondii (Fig. 6A), intact parasites were incubated with anti-SAG1 antibodies, specific for the major membrane protein of T. gondii (25), and diluted tears of five individuals. While the anti-SAG1 antibody gave a homogeneous fluorescent pattern contouring all parasites, IgA antibodies in tears gave a coarse fluorescence of most parasites and a homogeneous fluorescence of a minority (Fig. 6B, using sample V4). There were differences in fluorescence patterns between tear samples tested; sample V1 only displayed a coarse pattern, weaker and more apically localized (not shown). Two other incubations did not give detectable fluorescence patterns. In general, fluorescence was only observed with tear samples showing brisk recognition of PDI.




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Fig. 6. Tear antibody staining patterns of intact parasites. (A) Diluted tear samples of two individuals were incubated twice with PBS (–) or intact parasites (+), as described earlier (17). The IgA staining pattern after the second extraction is shown. (B) Intact parasites were double stained by incubation with tears of V4, known to have a brisk anti-49-kDa titer, and a mAb specific for the major surface antigen of T. gondii, SAG1. Staining of parasites by IgA in tears and anti-SAG1 antibodies was analyzed using a laser scanning microscope. Tear IgA and anti-SAG1 patterns are superimposed in ‘Overlay’. Red in the tear anti-IgA–FITC and SAG1–Cy3 pictures indicated saturated pixels. In ‘Overlay’, green indicates predominant tear IgA staining, red indicates predominant SAG1 staining, and yellow indicates co-localization of tear IgA and SAG1 staining. Tears of V4 were diluted 25 times. Bar: 2 µm.

 
Although suggesting a possible function, surface staining of IgA does not help to distinguish between conventional and natural antibodies as conventional IgM (and most likely also sIgA) responses are directed against surface-exposed epitopes of antigens (29), while natural sIgA are capable of binding to the surface of bacteria (11) and, as shown here, protozoan parasites.


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In a recent study we identified T. gondii PDI as the major antigen recognized by tear IgA (18). Now we demonstrate that anti-PDI sIgA are present in different mucosal secretions (tears and milk) from adult healthy human subjects, and tears from young children and infants. It is highly unlikely that these infants were exposed to T. gondii when nursed and there were no symptoms of an acquired infection of the mothers during gestation. Previous findings already pointed out that there is no relation between the presence of these IgA antibodies in tears and chronic infection by T. gondii, i.e. no correlation with presence or absence of T. gondii antibodies in serum. The fact that human milk also contains anti-PDI antibodies confirms that human milk is also a rich source of natural sIgA antibodies (10,16). Clearly, antibodies with specificity for PDI form part of the sIgA repertoire responsible for the primary protection of nursed infants and probably remain present in tears throughout life (17), stressing their importance.

PDI was one of the few antigens recognized by lacrimal IgA in infants and adults. The paucity of antigens recognized by natural antibodies in infants as compared to older children and adults has been observed previously in natural IgM repertoires (30). Apparently IgA anti-PDI antibodies are secreted by a select group of B cells naturally activated early in MIS development. As anti-PDI antibodies are continuously secreted irrespective of age (17), these B cells apparently remain present throughout life—a property shared with natural IgM-secreting B cells in mice (30). Remarkably, three siblings had similar anti-toxoplasma IgA staining patterns, including anti-PDI. Although autoimmune repertoires of natural IgM antibodies in sera of mice within a strain are almost identical, the autoimmune repertoires of congenic mice already differ (31). More research is required to determine the immunological framework responsible for identical patterns in siblings.

Detailed analysis of amino acid sequences recognized by anti-PDI antibodies showed at least two conserved regions involved, including a thioredoxin-like domain of PDI. This domain is highly conserved among PDI (19,32,33) (Fig. 4), suggesting that anti-T. gondii PDI IgA antibodies could react with PDI from other species. This was demonstrated with extracts of the closely related P. falciparum and cultured human cells. The other region of 8 amino acids usually contains at least six conserved residues, the VKVVVxxN motif. This region is conserved between T. gondii and human PDI sequences (Fig. 4). The conformational flexibility of natural antibodies could allow cross-reactivity with PDI that have moderately diverged (34,35). That conserved regions within a conserved protein are recognized, allowing interspecies cross-reactivity, establishes these anti-PDI antibodies as so-called natural antibodies and exponents of the innate immune system (see Fig. 5). In contrast, forced immunization with recombinant T. gondii PDI did not generate antibodies that are capable of cross-reaction, which indicates that these antibodies are primarily directed against species-specific regions of PDI (‘B’ regions, Fig. 4).

Table 3 summarizes our findings and those obtained by others regarding characteristics of natural antibodies directed against protein antigens. Although analysis of polyreactivity has been used convincingly to characterize sets of antibodies (10), this is the first report in which delineation of epitopes has been used for characterization of a particular (natural) antibody.


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Table 3. Are the anti-PDI IgA conventional or natural antibodies?
 
PDI is a multi-functional enzyme. It belongs to the superfamily of thioredoxins, functioning primarily as an endoplasmic reticulum oxidoreductase of eukaryotic cells. It facilitates de novo formation of disulfide bridges between cysteines (oxidation) and/or rearranges existing disulfide bridges (isomerization) in secretory and cell-surface proteins (19). All major surface antigens of T. gondii, SAG1-, SAG3- and SAG1-related proteins have high numbers of intramolecular disulfide bonds (25), which might explain the high expression level of T. gondii PDI.

Although its intracellular localization suggests that PDI is unavailable for binding by sIgA antibodies in human mucosal secretions, there are indications that PDI has functions apart from its role in the endoplasmic reticulum. Despite its endoplasmic reticulum retention signal, PDI is found on the surface of several cell types: on thyroid cells, where it is involved in shedding of the thyroid stimulating hormone receptor ectodomain (36); on platelets, where PDI mediates adhesion to integrin (37); on lymphocytes, involved in adhesion of HIV (38); and on hepatocytes (39). Our results demonstrate that T. gondii expresses PDI on its cell surface as well. Like PDI on the membrane of human cells, T. gondii PDI may contribute to adhesion processes, e.g. to host cells during infection, being involved in maintenance of structure and function of surface antigens, and/or the concomitant shedding of surface antigens, such as SAG1 (40). Importantly, this would make mucosal anti-PDI IgA function against T. gondii in the innate, first-line defense immune reaction. Although there are strong indications that tear IgA also recognizes P. falciparum PDI, it is not known whether PDI is exposed on the surface of this parasite as well.

Natural antibodies are usually cross-reactive, and recognize non-self antigens of pathogens and self-related antigens (2,8,10,34), like the anti-PDI antibodies described here. It is possible that the anti-PDI antibodies may be selected for their ability to bind to self-PDI, as both the thioredoxin-like domain and the VKVVVxxN motif are conserved between PDI. There are several models to explain natural antibody repertoire selection (30,31,41), but only one seems compatible with our results: anti-PDI sIgA could represent the evolutionary conserved germline repertoire of antibodies (10,30) and form part of the ‘natural immune memory’ (42,43) or ‘immunological homunculus’ (31). These antibodies might be selected for their ability to bind to evolutionary conserved self-antigens having the potential to be (ab)used by pathogens to adhere to cells and initiate infection, e.g. as PDI by HIV in adherence to lymphocytes (38). This implies that secretion of anti-PDI sIgA could be due to their ability to bind to both ‘self’ and ‘non-self’ PDI, including T. gondii PDI. The production of these antibodies appears to be largely confined to mucosal surfaces where dimeric IgA is rapidly secreted, thereby restricting their autoreactive potential.

T. gondii PDI was identified as a major target for naturally occurring human IgA antibodies secreted at mucosal sites. The epitopes on PDI recognized by these natural antibodies are evolutionarily conserved, suggesting they have evolved to participate in the innate immunity against protozoan parasites and perhaps other eukaryotic pathogens as well. Whether PDI can be regarded as a primitive invasion protein that can be neutralized by primitive (natural) IgA remains to be investigated. Our results suggest that the human lacrimal gland, along with the mammary gland, is one of the few mucosal sites with predominant secretion of natural antibodies. This should be confirmed by detailed analysis of plasma cells present at these sites.


    Abbreviations
 
IB—immunoblot

IF—immunofluorescence

MIS—mucosal immune system

MLN—mesenteric lymph node

PDI—protein disulfide isomerase

SAG—surface antigen

sIgA—secretory IgA

SC—secretory component

Ser—serum


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Brandtzaeg, P. 1995. Molecular and cellular aspects of the secretory immunoglobulin system. APMIS 103:1.[ISI][Medline]
  2. Bouvet, J. P. and Fischetti, V. A. 1999. Diversity of antibody-mediated immunity at the mucosal barrier. Infect. Immun. 67:2687.[Free Full Text]
  3. Mazanec, M. B., Nedrud, J. G., Kaetzel, C. S. and Lamm, M. E. 1993. A three-tiered view of the role of IgA in mucosal defense. Immunol. Today 14:430.[ISI][Medline]
  4. Ochsenbein, A. F. and Zinkernagel, R. M. 2000. Natural antibodies and complement link innate and acquired immunity. Immunol. Today 21:624.[ISI][Medline]
  5. Quiding-Jarbrink, M., Eriksson, K., Lakew, M., Butcher, E., Banchereau, J., Lazarovits, A., Holmgren, J. and Czerkinsky, C. 1996. Generalized and compartmentalized mucosal immune responses in humans: cellular and molecular aspects. In Kagnoff,M.F. and Kiyono,H., eds. Essentials of Mucosal Immunology, p. 477. Academic Press, San Diego, CA.
  6. McGhee, J. R. and Kiyono, H. 1993. New perspectives in vaccine development: mucosal immunity to infections. Infect. Agents Dis. 2:55.[ISI][Medline]
  7. Czerkinsky, C., Prince, S. J., Michalek, S. M., Jackson, S., Russell, M. W., Moldoveanu, Z., McGhee, J. R. and Mestecky, J. 1987. IgA antibody-producing cells in peripheral blood after antigen ingestion: evidence for a common mucosal immune system in humans. Proc. Natl Acad. Sci. USA 84:2449.[Abstract]
  8. Avrameas, S. 1991. Natural autoantibodies: from ‘horror autotoxicus’ to ‘gnothi seauton’. Immunol. Today 12:154.[ISI][Medline]
  9. Murakami, M. and Honjo, T. 1995. Involvement of B-1 cells in mucosal immunity and autoimmunity. Immunol. Today 16:534.[ISI][Medline]
  10. Quan, C. P., Berneman, A., Pires, R., Avrameas, S. and Bouvet, J. P. 1997. Natural polyreactive secretory immunoglobulin A autoantibodies as a possible barrier to infection in humans. Infect. Immun. 65:3997.[Abstract]
  11. Bos, N. A., Bun, J. C., Popma, S. H., Cebra, E. R., Deenen, G. J., van der Cammen, M. J., Kroese, F. G. and Cebra, J. J. 1996. Monoclonal immunoglobulin A derived from peritoneal B cells is encoded by both germ line and somatically mutated VH genes and is reactive with commensal bacteria. Infect. Immun. 64:616.[Abstract]
  12. Fischer, M. and Kuppers, R. 1998. Human IgA- and IgM-secreting intestinal plasma cells carry heavily mutated VH region genes. Eur. J. Immunol. 28:2971.[ISI][Medline]
  13. Holtmeier, W., Hennemann, A. and Caspary, W. F. 2000. IgA and IgM VH repertoires in human colon: evidence for clonally expanded B cells that are widely disseminated. Gastroenterology 119:1253.[ISI][Medline]
  14. Dunn-Walters, D. K., Isaacson, P. G. and Spencer, J. 1997. Sequence analysis of human IgVH genes indicates that ileal lamina propria plasma cells are derived from Peyer’s patches. Eur. J. Immunol. 27:463.[ISI][Medline]
  15. Dunn-Walters, D. K., Hackett, M., Boursier, L., Ciclitira, P. J., Morgan, P., Challacombe, S. J. and Spencer, J. 2000. Characteristics of human IgA and IgM genes used by plasma cells in the salivary gland resemble those used in duodenum but not those used in the spleen. J. Immunol. 164:1595.[Abstract/Free Full Text]
  16. Vassilev, T. L. and Veleva, K. V. 1996. Natural polyreactive IgA and IgM autoantibodies in human colostrum. Scand. J. Immunol. 44:535.[ISI][Medline]
  17. Meek, B., Klaren, V. N., van Haeringen, N. J., Kijlstra, A. and Peek, R. 2000. IgA antibodies to Toxoplasma gondii in human tears. Invest. Ophthalmol. Vis. Sci. 41:2584.[Abstract/Free Full Text]
  18. Meek, B., Back, J. W., Klaren, V. N., Speijer, D. and Peek, R. 2002. Protein disulfide isomerase of Toxoplasma gondii is targeted by mucosal IgA antibodies in humans. FEBS Lett. 522:104.[ISI][Medline]
  19. Frand, A. R., Cuozzo, J. W. and Kaiser, C. A. 2000. Pathways for protein disulphide bond formation. Trends Cell Biol. 10:203.[ISI][Medline]
  20. Beaman, M. H., McCabe, R. E., Wong, S. Y. and Remington, J. S. 1995. Toxoplasma gondii. In Mandell, G. L., Bennett, J. E. and Dolin, R., eds, Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases, p. 2455. Churchill Livingstone, New York.
  21. Kortbeek, L. M. 1999. Toxoplasmose in Nederland. Ned. Tijdschr. Klin. Chemie 24:65.
  22. Mack, D. G. and McLeod, R. 1992. Human Toxoplasma gondii-specific secretory immunoglobulin A reduces T. gondii infection of enterocytes in vitro. J. Clin. Invest. 90:2585.[ISI][Medline]
  23. Chardes, T., Bourguin, I., Mevelec, M. N., Dubremetz, J. F. and Bout, D. 1990. Antibody responses to Toxoplasma gondii in sera, intestinal secretions, and milk from orally infected mice and characterization of target antigens. Infect. Immun. 58:1240.[ISI][Medline]
  24. Coyle, P. K. and Sibony, P. A. 1986. Tear immunoglobulins measured by ELISA. Invest. Ophthalmol. Vis. Sci. 27:622.[Abstract]
  25. Manger, I. D., Hehl, A. B. and Boothroyd, J. C. 1998. The surface of Toxoplasma tachyzoites is dominated by a family of glycosylphosphatidylinositol-anchored antigens related to SAG1. Infect. Immun. 66:2237.[Abstract/Free Full Text]
  26. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[ISI][Medline]
  27. Sambrook, J. and Russel, D. W. 2001. Molecular Cloning, 3 edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  28. Cole, M. F., Bryan, S., Evans, M. K., Pearce, C. L., Sheridan, M. J., Sura, P. A., Wientzen, R. L. and Bowden, G. H. 1999. Humoral immunity to commensal oral bacteria in human infants: salivary secretory immunoglobulin A antibodies reactive with Streptococcus mitis biovar 1, Streptococcus oralis, Streptococcus mutans, and Enterococcus faecalis during the first two years of life. Infect. Immun. 67:1878.[Abstract/Free Full Text]
  29. Abbas, A. K., Lichtman, A. H. and Pober, J. S. 2002. Cellular and Molecular Immunology, 4 edn. Saunders, Philadelphia, PA.
  30. Mouthon, L., Nobrega, A., Nicolas, N., Kaveri, S. V., Barreau, C., Coutinho, A. and Kazatchkine, M. D. 1995. Invariance and restriction toward a limited set of self-antigens characterize neonatal IgM antibody repertoires and prevail in autoreactive repertoires of healthy adults. Proc. Natl Acad. Sci. USA 92:3839.[Abstract/Free Full Text]
  31. Nobrega, A., Haury, M., Grandien, A., Malanchere, E., Sundblad, A. and Coutinho, A. 1993. Global analysis of antibody repertoires. II. Evidence for specificity, self-selection and the immunological ‘homunculus’ of antibodies in normal serum. Eur. J. Immunol. 23:2851.[ISI][Medline]
  32. Florent, I., Mouray, E., Dali, A. F., Drobecq, H., Girault, S., Schrevel, J., Sergheraert, C. and Grellier, P. 2000. Cloning of Plasmodium falciparum protein disulfide isomerase homologue by affinity purification using the antiplasmodial inhibitor 1,4-bis[3-[N-(cyclohexyl methyl)amino]propyl]piperazine. FEBS Lett. 484:246.[ISI][Medline]
  33. Freedman, R. B., Hirst, T. R. and Tuite, M. F. 1994. Protein disulphide isomerase: building bridges in protein folding. Trends Biochem. Sci. 19:331.[ISI][Medline]
  34. Bouvet, J. P. and Dighiero, G. 2000. Cross-reactivity and polyreactivity: the two sides of a coin. Immunol. Today 21:411.[ISI][Medline]
  35. Wedemayer, G. J., Patten, P. A., Wang, L. H., Schultz, P. G. and Stevens, R. C. 1997. Structural insights into the evolution of an antibody combining site. Science 276:1665.[Abstract/Free Full Text]
  36. Delom, F., Mallet, B., Carayon, P. and Lejeune, P. J. 2001. Role of extracellular molecular chaperones in the folding of oxidized proteins. Refolding of colloidal thyroglobulin by protein disulfide isomerase and immunoglobulin heavy chain-binding protein. J. Biol. Chem. 276:21337–21342.[Abstract/Free Full Text]
  37. Lahav, J., Gofer-Dadosh, N., Luboshitz, J., Hess, O. and Shaklai, M. 2000. Protein disulfide isomerase mediates integrin-dependent adhesion. FEBS Lett. 475:89.[ISI][Medline]
  38. Ryser, H. J., Levy, E. M., Mandel, R. and DiSciullo, G. J. 1994. Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus–receptor interaction. Proc. Natl Acad. Sci. USA 91:4559.[Abstract]
  39. Terada, K., Manchikalapudi, P., Noiva, R., Jauregui, H. O., Stockert, R. J. and Schilsky, M. L. 1995. Secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes. J. Biol. Chem. 270:20410.[Abstract/Free Full Text]
  40. Dubremetz, J. F., Rodriguez, C. and Ferreira, E. 1985. Toxoplasma gondii: redistribution of monoclonal antibodies on tachyzoites during host cell invasion. Exp. Parasitol. 59:24.[ISI][Medline]
  41. Cohen, I. R. and Young, D. B. 1991. Autoimmunity, microbial immunity and the immunological homunculus. Immunol. Today 12:105.[ISI][Medline]
  42. Martin, F. and Kearney, J. F. 2000. B-cell subsets and the mature preimmune repertoire. Marginal zone and B1 B cells as part of a ‘natural immune memory’. Immunol Rev. 175:70.
  43. Berland, R. and Wortis, H. H. 2002. Origins and functions of B-1 cells with notes on the role of CD5. Annu. Rev. Immunol 20:253.
  44. Lydyard, P. M., Quartey-Papafio, R., Broker, B., Mackenzie, L., Jouquan, J., Blaschek, M. A., Steele, J., Petrou, M., Collins, P., Isenberg, D. and Youinou, P. Y. 1990. The antibody repertoire of early human B cells. I. High frequency of autoreactivity and polyreactivity. Scand. J. Immunol. 31:33.[ISI][Medline]
  45. Guilbert, B., Dighiero, G. and Avrameas, S. 1982. Naturally occurring antibodies against nine common antigens in human sera. I. Detection, isolation and characterization. J. Immunol. 128:2779.
  46. Dighiero, G., Guilbert, B., Fermand, J. P., Lymberi, P., Danon, F. and Avrameas, S. 1983. Thirty-six human monoclonal immunoglobulins with antibody activity against cytoskeleton proteins, thyroglobulin, and native DNA: immunologic studies and clinical correlations. Blood 62:264.
  47. Weksler, M. E. 2000. Changes in the B-cell repertoire with age. Vaccine 18:1624.
  48. Ferrari, D. M. and Soling, H. D. 1999. The protein disulphide-isomerase family: unravelling a string of folds. Biochem. J. 339:1.[ISI][Medline]