1 Department of Clinical and Experimental Medicine, University of Verona, 37134 Verona, Italy
2 DIMES, Department of Experimental Medicine, and
3 Department of Clinical and Experimental Oncology, University of Genova, 16132 Genova Italy
4 CBA/ABC Advanced Biotechnology Center, 16132 Genova, Italy
5 Institute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland
Correspondence to: A. Puccetti, Unità di Citologia MolecolareUnità Monoclonali B3, Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova, Italy
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Abstract |
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Keywords: autoantigens, glycine-rich protein, random peptide library
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Introduction |
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Attempts to characterize autoantigenic epitopes in different autoimmune diseases have been made possible using random peptide phage libraries which allow the identification of ligands for disease-specific antibodies whether the antigen is known or not (47). This approach identifies linear, conformational as well as mimic epitopes, and it has been used to screen Ig fractions obtained from patients with multiple sclerosis (8), chronic immune thrombocytopenic purpura (9), type 1 diabetes mellitus (10), systemic lupus erythematosus (SLE) (11) and rheumatoid arthritis (RA) (1214). One of the selected peptides identified by Ig from RA patients (12) showed homology with cereal glycine-rich cell wall proteins (GRP) and with the EpsteinBarr virus nuclear (EBV) antigen-I (EBNA-I). Analysis of GRP 1.8 (15), a ubiquitous protein found in French bean and related species, shows the presence of GlyAla repeated sequences homologous to EBNA-I and cytokeratins. Many studies have shown the ability of anti-EBNA-I antibodies to cross-react with different autoantigens such as heterogeneous nuclear ribonucleoprotein (hnRNP) (16), cytokeratin, collagen and actin in RA (17,18); the cross-reactions can be inhibited with synthetic peptides containing the GlyAla repeat sequence, implying that the cross-reactivity is due to anti-GlyAla antibodies that recognize both the host proteins and the EBNA-I repeat. A 15 amino acid synthetic peptide corresponding to amino acids 436454 of GRP 1.8 and sharing homology with EBNA-I, fibrillar collagen and procollagen (Table 1) has been shown to induce humoral (14) and T cell responses in RA (19). Taken together, these data suggest that the autoimmune response in RA but also in other autoimmune diseases may not be as heterogeneous as originally thought. A possible consequence of such hypothesis is that the identification of widespread (auto)antigens may allow their use in the suppression of autoimmunity by oral administration. The suppression of autoimmunity by oral antigen administration may be a feasible therapeutic option (reviewed in 20) and the induction of oral tolerance can be a therapeutic strategy also in food allergy (reviewed in 21). Based on the observation that GlyAla repeated sequences are present in food proteins as well as in viruses and self-proteins, we decided to investigate the presence of anti-GRP antibodies in a large panel of sera from patients with autoimmune disorders. The 15 amino acid peptide derived from GRP was used to analyze human sera in a direct and competitive ELISA assay. Serum IgG antibodies directed against such peptide were detected in different percentages of several autoimmune disorders and in food allergy. The GRP peptide was able to elicit a specific T cell response: peripheral mononuclear cells (PBMC) derived from patients with different diseases proliferated to the peptide and antigen-specific T cell clones could be generated from such patients. These data suggest that (auto)immune responses can be triggered by protein epitopes with crucial amino acids homologous to self proteins.
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Methods |
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IgM rheumatoid factor was present in 25 of 32 RA patients and its level varied between 105 and 770 IU/ml (n.v. < 60 IU/ml).
All the patients' sera were tested for the presence of the following autoantibodies: anti-ssDNA antibodies, anti-keratin antibodies, anti-collagen II antibodies, anti-RNP antibodies. Anti-ssDNA antibodies were present in 37 of 37 SLE patients and in two of 32 RA patients, anti-keratin antibodies in seven of 32 RA patients and two in 12 of PsA patients, anti-collagen antibodies in eight of 32 RA patients and in one of 12 PsA patients, and anti-hnRNP antibodies in eight of 37 SLE patients. The sera of patients affected by CIU, food allergy and aero-allergy were negative for the autoantibody specificities studied.
Peptide synthesis and recombinant GRP proteins
The synthetic peptides (GRP peptide: GGYGDGGAHGGGYGG and the irrelevant control peptide: ALYPSSVGQPFQGAP) were obtained by solid-phase synthesis using Fmoc-protected amino acids according to the method of Merrifield as modified by Atherton (25), and were purified by gel filtration. Recombinant GRP proteins with or without the sequence corresponding to the GRP peptide were expressed in Escherichia coli and purified according to Ryser et al. (15).
mAb and reagents
The purified mAb anti-CD56 (Leu19, IgG1), anti-CD8 (Leu2a, IgG1), anti-CD4 (Leu3a, IgG1), anti-CD3 (Leu4, IgG1) and anti-CD19 (Leu12, IgG1) were from Becton Dickinson (San Jose, CA). The purified mAb anti-CD16 (KD1, IgG2a) was a kind gift of Professor L. Moretta (Genoa, Italy). The class II specific mAb DC1-12 was a kind gift of Professor R. Accolla (Genoa, Italy). Phycoerythrin (PE)- or FITC-conjugated goat anti-mouse antisera were from Southern Biotechnology Associates (Birmingham, AL). Human keratin, collagen type II and actin were purchased from Sigma (St Louis, MO).
Affinity purification of IgG anti-peptide and anti-keratin antibodies
Serum IgG was obtained by affinity purification using a Protein ASepharose column (Pharmacia, Uppsala, Sweden). The synthetic peptides or human keratin (5 mg antigen/g dried Sepharose powder) were coupled to Sepharose CL4B (Pharmacia), according to the manufacturer's instructions. Affinity-purified IgG samples diluted in PBS were applied to the column. Bound IgG was eluted with 0.1 M glycine (pH 2.5) and dialyzed against PBS.
ELISA assay
The synthetic peptides and the recombinant proteins were used at a concentration of 20 µg/ml in PBS to coat polystyrene plates (Nunc, Roskilde, Denmark). After blocking with 5% dry non-fat milk in PBS, the antibodies diluted in 2.5% dry non-fat milk and 0.05% Tween in PBS were added and incubated for 4 h. The plates were then washed and alkaline phosphatase-conjugated goat anti-human IgG (F(ab')2 fragment) or anti-mouse IgG or IgM (Sigma) were added and incubated overnight at 4°C. After washings, the bound enzymatic activity was measured with p-nitrophenylphosphate (Sigma). Each antibody preparation was tested on a control plate not coated with the antigen. This non-specific binding never exceeded 10% of the specific binding (e.g. to the antigen coated plate). For competitive assays the amount of antibody that gave 50% of the maximum binding to the antigen on the solid phase was preincubated with different amounts of competitors or buffer for 1 h at 37°C and then transferred to the antigen-coated plates. The assay was then carried on as the direct binding assay. In the ELISA assay for the detection of serum antibodies directed against the peptide or the recombinant GRP protein containing the peptide sequence of interest, 25 sera diluted 1: 100 from normal age- and sex-matched subjects were used as control group. Optical density values higher than the mean + 3 SD of each serum dilution of the control group (OD > 70 for the GRP peptide and OD > 85 for the recombinant protein) were considered positive.
The direct and competitive ELISA for human keratin, collagen type II and actin has been described (24). The ELISA assay for EBNA-I was performed using a commercially available kit (Sigma).
Immunization of BALB/c mice
The synthetic peptide was coupled to the carrier protein keyhole limpet hemocyanin (KLH) and emulsified in Freund's adjuvant. Mice were injected 5 times (the first time in complete Freund's adjuvant, the other times in incomplete adjuvant) at the base of the tail at 15 day intervals. The animals were bled 7 days after the last injection and the sera tested in ELISA. Control animals were injected with adjuvant alone, keyhole limpet hemocyanin (KLH) alone or coupled with the irrelevant peptide.
Generation of CD4+-specific T cells clones and flow cytofluorimetric analysis
PBMC derived from patients were isolated on Ficoll-Hypaque gradient and cells were then incubated with peptide at 20 µg/ml in 96 U-bottomed microplates in complete medium. After 10 days of culture in absence of rIL-2 the cells were incubated with a mixture of anti-CD16 (KD1), anti-CD56 (Leu19) and anti-CD8 (Leu2a) mAb and purified by immunodepletion using goat anti-mouse Ig coated with magnetic beads (Unipath, Milan, Italy) (26). Viable cells were cloned under limiting dilution in the presence of irradiated peripheral blood lymphocytes as feeder cells in complete medium and of exogenous rIL-2 (Cetus, Emeryville, CA) as described for T cell cloning (27).
Cells were stained with the appropriate mAb followed by fluoresceinated goat anti-mouse Ig (28); control aliquots were stained with the fluoresceinated reagent alone. All samples were analyzed on a flow cytometer FACSort (Becton Dickinson) gated to exclude non-viable T cells.
Human mAb
EpsteinBarr virus-transformed cell lines were derived from selected individuals according to standard procedures (29) and were cloned in soft agar; the resulting Ig-producing clones were tested for antigen-binding activity by ELISA assay.
Proliferation assay
PBMC or highly purified CD4+ clones were cultured for 3 days in complete medium in 96-well U-bottom microplates (5x105 cells/well) with irradiated autologous PBMC or B-EBV-transformed cell lines (5x105 cells/well). The following stimuli were used: GRP peptide (20 µg/ml), an irrelevant peptide (20 µg/ml) and rIL-2 (20 U/ml) as positive control. Cells were then pulsed with 20 µCi [3H]thymidine and incubated for an additional 18 h at 37°C. Results are expressed in c.p.m.x103 of the mean ± SD of triplicate samples of two different tests for each patient.
Identification of the phenotype of individual clones.
The Th subsets of the clones obtained was analyzed by flow cytometric assessment of intracytoplasmic cytokine content (reviewed in 30). Cells were stimulated with 25 ng/ml phorbol 12-myristate 13-acetate (Sigma) plus 1 µg/ml ionomycin (Sigma) for 4 h in the presence of 10 µg/ml Brefeldin A (Sigma) (31) and were subsequently fixed with PBS containing 4% (v/v) paraformaldehyde and permeabilized in PBS/saponin buffer (Sigma). Directly conjugated monoclonal anti-cytokine antibodies specific for IL-2, IFN-, 1L-4, IL-5 and IL-10 were used to identify the cytokines produced [FITC-conjugated anti-IFN-
and R-PE-conjugated anti-IL-2 and -IL-4, from Becton Dickinson; R-PE-anti-IL-5 and -IL-10, from PharMingen (San Diego, CA)]. Specificity controls were performed using isotypical mAb (IgG2aFITC and IgG1R-PE, both from Becton Dickinson). At least 30,000 events were acquired by FACScan flow cytometer equipped with an argon ion laser (488 nm) and CellQuest software (Becton Dickinson).
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Results |
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Anti-GRP IgE antibodies were not detected in any patients' group.
These data suggest that anti-GRP peptide IgG antibodies are widely present in the serum of patients with autoimmune disorders and with food allergy.
Serum anti-peptide antibodies specifically recognize the native recombinant protein
Prokaryotic expression plasmids encoding truncated forms of the GRP were expressed in E. coli (15). SDSPAGE analysis of lysates of E. coli expressing these truncated forms demonstrated that each lysate contained an additional protein of the expected molecular mass. The different recombinant proteins were then purified and used in an ELISA assay to confirm the reactivity of the sera with the peptide. Fifteen patients' sera containing serum antibodies against the GRP peptide as well as normal controls' sera were tested on the various forms of recombinant proteins. All the 15 sera reacted with the recombinant protein (GST1fGRPC) containing the insert corresponding to the GRP peptide sequence, but not with the other truncated versions of the molecule. Similarly sera which did not recognize the GRP peptide did not react with the recombinant protein. Normal human sera did not recognize the recombinant protein (data not shown). In a competitive immunoassay the liquid-phase GRP peptide could displace the binding of serum Ig from the recombinant protein on the solid phase (Fig. 1B), further confirming the specificity of such antibody interaction. The data obtained indicate that serum antibodies are able to recognize the GRP peptide sequence even in the context of a larger molecule such as the GRP recombinant protein. This observation is important because it may constitute the basis for a wide cross-reactivity of antibodies directed against the GRP peptide.
Serum anti-peptide antibodies cross-react with other autoantigens
To further characterize the fine specificity of the binding of serum anti-GRP peptide antibodies we isolated the IgG anti-peptide component from the sera of five different patients by affinity chromatography using a peptideSepharose column. Anti-GRP peptide antibodies affinity purified from five normal donors were used as control. Anti-GRP peptide antibodies isolated from the patients' sera recognized the peptide in both direct and competitive ELISA, and they also reacted with the recombinant protein (data not shown).
These antibody preparations were then tested for their ability to recognize other autoantigens such as human keratin, collagen type II, actin, EBNA-I. As shown in Table 3, the anti-GRP peptide reacted with different autoantigens and the affinity of the interaction varied within the different serum samples. On the contrary anti-GRP peptide antibodies purified from the serum of normal subjects showed a low-affinity interaction with the GRP peptide and did not cross-react with any of the autoantigens tested. Moreover 20 times more serum was needed to purify the same amount of anti-peptide Ig when compared to the patients' sera, indicating that such anti-peptide antibody population was much less represented in the sera of the healthy donors as compared to the patients studied. Figure 2
(A and B) shows a cross-inhibition experiment in which the binding of two anti-GRP peptide antibody preparations to solid-phase keratin is cross-inhibited by the GRP peptide but not by an irrelevant control peptide.
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These results indicate that anti-GRP peptide antibodies isolated from the sera of autoimmune patients are able to cross-react with autoantigens including keratin, collagen and EBNA-I. This cross-reactivity is not shown by anti-control peptide antibodies obtained from the same subjects.
In a separate set of experiments anti-keratin antibodies were affinity purified from the serum of patients GC and SP using a keratinSepharose column. The purified antibody preparations recognized human keratin in direct and competitive ELISA (data not shown). Such anti-keratin antibody populations were used to perform a cross-inhibition experiments in which the binding of these antibodies to solid-phase keratin is competed by keratin and GRP peptide. As shown in Fig. 2(C and D), the binding of these antibodies to keratin is completely inhibited by keratin and only partially by GRP peptide. These data indicate that anti-GRP antibodies are able to cross-react with autoantigens (keratin, actin, EBNA-I), whereas not all the antibodies directed against such autoantigens are able to recognize the GRP sequence.
These results indicate that the peptide studied identifies a shared epitope widely expressed in different diseases and that antibodies against such peptide recognize different autoantigen targets.
EBV-transformed cell clones producing anti-GRP antibodies
Six EBV-transformed cell clones obtained from patients PG, GC and SP produced anti-GRP peptide antibodies able to recognize keratin, actin and EBNA-I. An example of this behavior is given in Fig. 3. These results confirm at the clonal level our previous observations that anti-GRP antibodies produced in subjects with autoimmune disorders are able to cross-react with several autoantigens and this cross-reactivity may be relevant in the pathogenesis of the disease.
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One hundred and fifty antigen-specific T cell clones were derived from the PBMC of the 11 patients studied. The clonal efficiency of the clonal procedures was 4.82% (SD ± 1.1). We then chose to characterize 38 clones on the basis of their growth capability; all the clones proved to be CD4+ by FACS analysis. The antigen specificity of the 38 clones was then evaluated using the GRP peptide and EBV-transformed autologous B lymphocytes or autologous PBMC as antigen-presenting cells. Twenty-five T cell clones showed a specific proliferative response to the GRP peptide, but not to an irrelevant control peptide. Of the 25 peptide-specific T cell clones, six were derived from patient SP, four from patient CA, two from patients SE, CG, VM, LM, PG, LoM, one from each of ZP, CoL, BS. The specificity of the proliferative response of the CD4+ T cell clones obtained from the patients studied was confirmed by the following lines of evidence: (i) all the anti-GRP T cell clones did not proliferate in the presence of the GRP peptide when heterologous irradiated EBV cells or heterologous PBMC were used as antigen-presenting cells and (ii) preincubation of the CD4+ anti-GRP peptide clones with the class II-specific mAb D1-12 abolished the proliferative response of these clones to the GRP peptide (data not shown). Figure 4 shows the proliferative response of representative clones derived from different patients.
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Taken together, these results suggest that a specific T cell response against the GRP peptide can be elicited in patients affected by different autoimmune diseases.
Intracytoplasmic cytokine profile of the T cell clones studied
In order to evaluate the Th subset of the peptide-specific T cell clones, we performed intracellular cytokine staining by FACS analysis. The 18 clones analyzed did not show a Th1 or Th2 cytokine profile, since IFN-, IL-2 and IL-4 were simultaneously present (data not shown). These data suggest that GRP peptide-specific T cell clones belong to the Th0 subset.
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Discussion |
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The finding that anti-GRP IgG antibodies are present at high frequency in the sera of autoimmune and food allergic individuals was further confirmed by the generation of human mAb from EBV-transformed lymphocytes belonging to representative patients. The anti-GRP peptide reactivity was present at high frequency in a panel of randomly selected EBV clones. Similarly to the serum counterpart, these antibodies were able to cross-react with several autoantigens. These results again support an important role for the GRP sequence in priming an autoimmune response. This hypothesis was further substantiated by experiments in which the 15 amino acid GRP peptide was coupled to a carrier protein and its ability to elicit an autoimmune response in normal animals (BALB/c mice) was analyzed. Interestingly mice displaying a strong anti-peptide response produced antibodies able to bind the GRP peptide, and to cross-react with several autoantigens including keratin, collagen II and EBNA-I.
In accordance to the humoral response, T cell responses can be elicited by the GRP peptide in different patients and in several disease conditions. A T cell response against the GRP peptide has been already described in the synovial fluid lymphocytes of patients with juvenile RA and GRP peptide specific CD4+ T cell clones have been obtained from the synovial fluid of these patients (19). Interestingly the GRP peptide is able to induce proliferation of PBMC isolated from patients with different diseases, whereas normal subjects do not proliferate to the peptide.
To further analyze the T cell response to the peptide, a panel of CD4+ peptide-specific T cell clones was derived from PBMC of patients with different disease conditions. CD4+ T cell clones specific for the GRP peptide could not be derived from the PBMC of normal healthy donors. Peptide-specific CD4+ T cell clones may either be the consequence of a previous activation and expansion by exogenous antigens, e.g. in the gut mucosa or more likely derive from normally present autoreactive T cell subsets (3). Independently from the mechanism involved in the generation of such CD4+ T cell clones, these cells may be able to provide a T cell help to B cells for the generation of an IgG response towards the peptide, which will result in the production of large amounts of cross-reacting anti-peptide antibodies. The presence of the same structural motif in the different autoantigens may account for the promiscuity of such autoantibody response which seems to be a common feature of the immune response in different autoimmune disorders. These results suggest that the immune response in autoimmune diseases may not be as heterogeneous as originally thought. In this regard it is possible to view the autoimmune response as an oligoclonal expansion of a rather limited number of T cell subsets which share the capability of responding to a particular amino acid sequence. The process may result in the stimulation of cells able to deliver T cell help to a great number of B cell clones. Thus, a large number of antibodies specific for different autoantigenic targets may arise as a response to a relatively small number of amino acid sequences, probably due to a mechanism of molecular mimicry.
Interestingly, a B and T cell response to the GRP peptide can also be found in patients with food allergy. The absence of anti-GRP-specific IgE antibodies in patients with food allergy suggests the possibility of a non-IgE-mediated immune response to particular food-derived antigens which can be able either to block or to mediate histamine release from basophils and mast cells.
The type of immune response elicited may be determined by the genetic background: the TCRpeptideMHC interaction can control the direction of the functional immune response, and MHC linkage to polarized Th1-type and Th2-type immune responses has now been reported for several antigens and peptides (3238). The analysis of some of our peptide-specific T cell clones derived from peripheral blood of patients with different diseases revealed a predominant Th0 phenotype. We can hypothesize either that the GRP peptide is unable to induce a dominant Th1 or Th2 cytokine response, or that this peptide-specific CD4+ subset found in the peripheral blood may switch to a Th1 or Th2 cytokine response at sites of autoimmune inflammation.
Several lines of evidence suggest that the induction of oral tolerance by orally administered antigens has potential therapeutic applications for the treatment of autoimmmune and food-allergic diseases (20,21). In this regard the finding that the same peptide epitope can induce an immune response in different diseases may have important practical implications. The identification of widespread peptide antigens may suggest their potential utilization in the suppression of the immune response by oral administration. This therapeutic strategy has so far been hampered mainly by the limited knowledge of disease relevant antigens.
Random peptide library technology is a powerful tool for identifying potentially pathologically relevant peptide antigens in different disease conditions. Using this approach it is possible to dissect common features at the amino acid level in individuals affected by the same or by different diseases. This could allow the identification of peptide sequences recognized at high frequency in certain conditions: the GRP peptide represents an example of an antigenic peptide sequence able to prime a B and T cell immune response in different and apparently unrelated diseases.
Finally, the finding of a common peptide epitope able to elicit an immune response in patients with food allergy and different autoimmune disorders give rise to the question of a possible link between food antigens, gut mucosa and systemic immune response. In the past few years several groups have studied the role of dietary manipulation in RA (3941), juvenile RA (42) and vasculitis (43), reaching conflicting results. T cell clones specific for particular food antigen epitopes may arise in the gut mucosa and be recruited to particular sites, such as joints, where they proliferate in response to homologous peptides derived from synovial proteins, following local inflammation and up-regulation of MHC molecules. The release of additional self-antigens and/or epitope spreading can lead to a chronic self-perpetuating process of organ inflammation and destruction.
In conclusion, our data suggest that phylogenetically highly conserved epitopes in plants, viruses and humans may be responsible for an autoimmune response in susceptible individuals.
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Acknowledgments |
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Abbreviations |
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CIU chronic idiopathic urticaria |
CPI chronic parvovirus infection |
EBNA-I EpsteinBarr nuclear antigen I |
EBV EpsteinBarr virus |
GRP glycine-rich cell wall protein |
hnRNP heterogeneous nuclear ribonucleoprotein |
KLH keyhole limpet hemocyanin |
PBMC peripheral blood mononuclear cells |
PE phycoerythrin |
PsA psoriatic arthritis |
RA rheumatoid arthritis |
SLE systemic lupus erythematosus |
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Notes |
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Transmitting editor: L. Moretta
Received 1 October 1999, accepted 25 January 2000.
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References |
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