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Low HEMA Conjugation Induces High Autoantibody Titer in Mice

¹ Section for Oral Immunology,
² Section for Endodontology, Faculty of Odontology, and
³ Department of Rheumatology, Faculty of Medicine, The Sahlgrenska Academy, Göteborg University, Box 450, SE 405 30 Göteborg, Sweden;

Correspondence: ^* corresponding author, Elisabeth.Sandberg{at}odontologi.gu.se

	ABSTRACT

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2-hydroxyethylmethacrylate (HEMA) is a known causal agent of hypersensitivity to resin composites. We have reported that immunization with HEMA conjugated to mouse serum albumin (MSA) induces an autoantibody response in mice. In this study, we investigated both the activity and the avidity of autoantibodies induced by immunization with various HEMA conjugations to MSA. Female Balb/c mice were given MSA carrying 3, 7, 15, or 22 HEMA molecules. Antigen-specific IgG and IgE antibodies were determined by ELISA, and average antibody avidity by thiocyanate dissociation. Immunization with MSA carrying the lowest number of HEMA molecules induced a significantly higher IgG and IgE anti-MSA autoantibody response, with significantly higher IgG antibody avidity, than did the more heavily conjugated preparations. The results suggest that the lower the degree of HEMA conjugation to self-protein, the higher the risk for autoantibody production to the carrier protein. These findings suggest a mechanism of potential relevance in humans.

Key Words: acrylates • autoantibody • autoimmunity • biomaterials • dental material

	INTRODUCTION

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Constituents in resin composites—e.g., 2-hydroxyethylmethacrylate (HEMA)—have been implicated in hypersensitivity reactions, including allergic contact dermatitis (Hensten-Pettersen, 1998; Kanerva et al., 1999; Wrangsjö et al., 2001; Alanko et al., 2004). This condition is regarded as a T-cell-mediated immune response induced by chemicals with the ability to penetrate skin or mucosal barriers and to react with host proteins, recently reviewed by Martin (2004). We have previously shown, in vitro, that HEMA can bind to host protein (Sandberg et al., 2002).

Besides hypersensitivity reactions, another possible consequence of HEMA exposure and its binding to self-protein is the production of autoantibodies. By exposure to proteins with similarities to self-proteins—e.g., viral proteins or protein homologues from other species—tolerance toward self can be broken (Oldstone, 1998; Bunder et al., 2004). In our previous report, mice immunized with mouse serum albumin (MSA), conjugated in vitro with HEMA, developed not only antibodies to the HEMA-modified conjugate, but also antibodies to the native carrier protein (Sandberg et al., 2002). While autoimmune disease may not necessarily follow, most autoimmune disorders are associated with the presence of autoantibodies. They can be significant for disease manifestation, as, for example, in myasthenia gravis, atopic dermatitis, and bullous pemphigoid (Natter et al., 1998; Hoch et al., 2001; Thoma-Uszynski et al., 2004), or they may be seemingly harmless but useful as diagnostic markers (Song et al., 2003). Possibly, the function and the location of the autoantigen are important parameters for autoantibodies to cause disease.

The development of an autoantibody response to a chemically modified self-protein might be dependent on the conjugation degree. A study of mouse lysozyme as a model protein found that the response to the self-protein was enhanced when the degree of modification was small (Tsujihata et al., 2000, 2001). In our earlier study, we observed that, at physiological pH, the degree of conjugation of the chemical to protein was low, implying that only a few HEMA molecules are likely to become attached in vivo (Sandberg et al., 2002). Another important aspect of antibody responses in general is the strength of the interaction between antigen and antibody, i.e., the avidity, since biological effects are greatly influenced by the properties of the immune complexes formed (Steward and Steensgaard, 1983).

Given our observed immunological effects of HEMA, the purpose of the present study was to explore the influence of different degrees of conjugation of HEMA to a self-protein on the subsequent antibody responses to the carrier.

	MATERIALS & METHODS

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Preparation of Mouse Serum Albumin
To avoid the problem with endotoxin contamination in commercially available substances, we prepared mouse serum albumin at our laboratory, using an FPLC system with 3 columns: Hiprep 16/10 Q FF (17-5190-01), HiPrep Sephacryl S-100 26/60 (17-1194-01), and HiTrap Protein G HP (17-0404-03) (Amersham Biosciences AB, Uppsala, Sweden). A 10-mL quantity of serum (Charles River, Sulzfeld, Germany) was mixed 1:1 with 20 mM Tris buffer (pH 8.0) and dialyzed for 24 hrs, then centrifuged at 4°C, 1000 g, for 10 min. The supernatant was filtered through a sterile 0.2-µm filter (Acrodisc^® Syringe Filter ref. 4454, Pall Gelman Sciences, Lund, Sweden), and passed over the ion exchange column in aliquots. Albumin was eluted by a salt-gradient: 20 mM Tris (pH 8.0) as start buffer, and 20 mM Tris with 1 M NaCl as eluting buffer. The fractions of interest were pooled and dialyzed against PBS for 48 hrs, with 2 changes, then concentrated on a filter device with a molecular cut-off of 30 kDa (Centriplus^®, Amicon no 4412, Millipore, Sundbyberg, Sweden), before being passed over the gel filtration column, coupled in series with the protein G column. The albumin peaked around 104 mL. The pool was concentrated and filtered through a sterile 0.2-µm filter. Analyses on SDS-PAGE and ELISA confirmed that the protein was albumin. In a Limulus amebocyte lysate assay, with a chromogenic end-point, aliquots were found to be negative regarding endotoxin content, with the limit of detection 0.006 EU. This analysis was carried out by the Dept. of Clinical Bacteriology, the Sahlgrenska Academy.

Conjugation of HEMA to Protein
Prior to incubation with MSA, HEMA (H8633, Sigma Chemical Co., St. Louis, MO, USA) was passed over a column (Aldrich No. 30631-2, Sigma-Aldrich) for removal of the inhibitor hydroquinone. Incubations were subsequently carried out at pH 8.1 in 0.1 M HCO₃ at 37°C. The reaction was stopped after 68 hrs by dialysis against PBS, pH 7.2. The preparations were filtered through a sterile 0.2-µm-sized filter before storage at –20°C. We derived the different conjugation degrees of 3, 7, or 15 HEMA molecules per albumin (MSA_H3, MSA_H7, and MSA_H15) by incubating HEMA in a molecular excess of 100, 1000, or 3000, respectively, to a protein concentration of 18.5 mg/mL. To reach the highest HEMA concentration (MSA_H22), we first concentrated the protein solution 5 times before adding HEMA gently to a molecular excess of 1000. Control MSA was treated identically, with incubation for 68 hrs in 0.1 M HCO₃, followed by dialysis. HEMA was furthermore similarly conjugated to ovalbumin (OVA), yielding a conjugation degree of 18%.

The average number of HEMA monomers bound to the protein was estimated based on the level of TNP conjugation, described in our previous report (Sandberg et al., 2002). In the calculations of the number of HEMA molecules bound, it was assumed that HEMA binds to the lysine residues, of which MSA contains 48 (Peters, 1996). An inhibition degree of, e.g., 6% would then indicate the attachment of 3 HEMA molecules per albumin molecule, on average.

Immunization Procedure
Female, 7- to 8-week-old Balb/c mice were used. Animals were immunized subcutaneously with a 25-µL PBS solution of 50 µg MSA, MSA_H3, MSA_H7, MSA_H15, or MSA_H22, respectively, emulsified in an equal volume of Freund’s incomplete adjuvant (FICA). One group was sham-immunized with PBS/FICA. Two similar booster injections were given after 2 and 6 wks. Seven days after the last booster, the animals were killed. Serum was taken and kept frozen until analysis. The protocol for the study was approved by the Göteborg Ethical Committee on Animal Experiments.

ELISA Assessments
IgG
Plates (Nunc immuno plate, Maxisorp^TM Kamstrup, Denmark) were coated overnight, at 4°C, with MSA, MSA_H, OVA, or OVA_H in PBS buffer, pH 7.2, before overnight incubation with serum samples. Next-day plates were incubated at room temperature, 2 hrs, with biotin-conjugated goat anti-mouse IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA), followed by extravidin-alkaline phosphatase (E2636 Sigma) for 2 hrs, then developed with para-nitrophenyl phosphate (S0942 Sigma), 1 mg/mL, dissolved in diethanolamine buffer, pH 9.8. Absorbance was read on a Spectra MAX 340 at 405 nm (Molecular Devices, Sunnyvale, CA, USA). A standard made of a pool of positive samples was run on each plate. Between incubations, wells were washed 3–4 times with PBS-Tween 20.

IgE
To avoid IgG antibody competition, we assessed the IgE anti-MSA antibody-specific portion from total IgE captured to the plates.

Plates were coated overnight with rat anti-mouse IgE (clone R35-72, BD Bioscience Pharmingen, Stockholm, Sweden) in PBS buffer, pH 7.2, then blocked with 0.5% casein for 30 min. Serum samples (starting dilution 1:50) and standard mouse IgE (clone IgE-3, BD Pharmingen), or a pool of positive sera, was incubated overnight, before incubation with biotinylated rat anti-mouse IgE (clone R35-118, BD Pharmingen) or biotin-conjugated MSA, for the assessment of both total and MSA-specific serum IgE, respectively. Plates were washed with PBS-Tween 20 between incubations and developed with extravidin-alkaline phosphatase and para-nitrophenyl phosphate, as described above.

We confirmed the specificity of the IgE ELISA by assessing IgE and IgG antibody activity following heat treatment of the serum at 56°C for 4 hrs. This treatment affected the IgE readings but not those for IgG.

Avidity
We estimated the average antibody avidity by measuring the degree of dissociation of antibodies bound to a solid-phase antigen when incubated with KSCN (Pullen et al., 1986). The analysis is similar to ELISA, except for one additional step. Subsequent to incubation with serum samples, plates were incubated for 15 min with KSCN at 6 concentrations, ranging from 0.1 to 3.2 M, in duplicate. Triplicate wells were incubated with PBS. After 6 PBS-Tween 20 washes, the analysis proceeded according to the ELISA protocol. An avidity index was calculated as the KSCN-concentration at which 50% of the antibody activity was eluted, compared with the highest activity obtained in wells incubated with the innocuous PBS.

	RESULTS

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Antibody Responses
IgG Antibodies to Native MSA
Nearly all animals immunized with HEMA-conjugated MSA produced antibodies to native MSA. Immunization with MSA_H3 resulted in the highest IgG autoantibody response (Fig. 1).

View larger version (10K): [in this window] [in a new window]   Figure 1. IgG antibody activity to native mouse serum albumin (MSA) in sera from animals immunized with MSA carrying various numbers of 2-hydroxyethylmethacrylate (HEMA) molecules: 3, 7, 15, or 22, respectively. Displayed are also control groups immunized with unconjugated MSA or adjuvant alone. IgG antibody activities are expressed in arbitrary ELISA units, as compared with a standard included in each of the assays. Dots denote 1 animal. Horizontal bars indicate median value. The autoantibody production was significantly higher in animals immunized with MSA carrying only 3 HEMA molecules (MSAH3), compared with those animals that were immunized with the other conjugation degrees (n = 8 for all groups, except for the one given MSAH22, where n = 7). The Mann-Whitney U-test was used for statistical comparison (* = p < 0.05, ** = p < 0.01, and *** = p < 0.005).

View larger version (12K): [in this window] [in a new window]   Figure 2. Total (A) and anti-MSA specific (B) antibody activity in serum from mice immunized with mouse serum albumin (MSA) carrying various numbers of 2-hydroxethylmethacrylate (HEMA) molecules: 3, 7, 15, or 22, respectively. Displayed are also control groups immunized with unconjugated MSA or adjuvant alone. Each dot represents 1 animal, and the horizontal bars indicate median values. (A) Significantly higher serum IgE concentration was found in animals immunized with MSA carrying only 3 HEMA molecules, compared with those immunized with MSA carrying 22 (n = 8 for all groups, except for the one given MSAH22, where n = 7). Statistical comparison was done by the Mann-Whitney U-test. (B) The specific IgE anti-MSA antibody activities in sera from animals treated as in (A). The antibody activities to native MSA are expressed as arbitrary ELISA units compared with a standard. Only animals immunized with MSAH3 displayed this antibody activity.

IgG Antibodies to HEMA Conjugated to OVA
We measured antibody activity to HEMA conjugated to OVA, to detect antibodies specific for HEMA, regardless of the carrier. Only the animals immunized with MSA_H15 and MSA_H22 gave a significant response to OVA_H when the anti-OVA antibody activity was subtracted (Fig. 3).

View larger version (10K): [in this window] [in a new window]   Figure 3. IgG antibody activity to 2-hydroxyethylmethacrylate (HEMA) when conjugated to a carrier that was not used in the immunizations: ovalbumin (OVA). Sera were taken from animals immunized with mouse serum albumin (MSA) carrying various numbers of (HEMA) molecules: 3, 7, 15, or 22, respectively. Displayed are also control groups immunized with unconjugated MSA or adjuvant alone. IgG antibody activities are expressed in arbitrary ELISA units as compared with a standard included in each of the assays. The IgG antibody activity to OVA has been subtracted. Each dot represents 1 animal, and the horizontal bars indicate median values (n = 8 for all groups, except for the one given MSAH22, where n = 7). Antibodies that could bind to HEMA, regardless of the carrier, were mainly induced by MSA carrying 15 or more HEMA molecules. The Mann Whitney U-test was used in statistical comparison between animals immunized with MSAH3 and those immunized with MSAH22.

View larger version (12K): [in this window] [in a new window]   Figure 4. Avidity index for antibodies induced in animals immunized with mouse serum albumin (MSA) carrying 3 or 15 2-hydroxyethylmethacrylate (HEMA) molecules, respectively. Serum was diluted, on average, 1:5000 for MSAH3 and 1:2000 for MSAH15 to yield a similar antibody activity. Wells were incubated with potassium thiocyanate (KSCN) at different concentrations, ranging from 0.1 to 3.2 M, in duplicate. Triplicate wells were incubated with PBS so that maximal activity could be assessed. The avidity is expressed as the KSCN concentration where 50% of the antibody activity is eluted compared with the activity in wells incubated with innocuous PBS. Each group consisted of 8 animals. One animal was omitted from the MSAH15 group, due to low anti-MSA antibody activity. Each dot represents 1 mouse, and the horizontal bars indicate median values. The Mann Whitney U-test was used in statistical comparison.

	DISCUSSION

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The observation that a slight modification of a self-protein may result in high potential to induce autoantibodies is in line with other reports. The substitution of 3 amino acids in mouse lysozyme induced a stronger autoantibody response than replacement of a whole peptide (Tsujihata et al., 2000). After immunization with MSA conjugated with maleyl on the lysine residues to more than 90%, no autoantibody production could be detected (Abraham et al., 1997). Our series of 6, 15, 32, or 46% of the lysine residues of MSA being occupied by HEMA showed that the production of autoantibodies seemed to decrease as the conjugation degree was increased.

A possible explanation for the induction of autoantibodies could be that B-cells specific for the native parts of the albumin molecule received help from T-cells specific for HEMA-modified peptides. The observation that a low degree of conjugation induced high autoantibody activity fits well with this concept, since more of the native surface would be left unchanged for binding by autoreactive B-cells. In contrast, at high conjugation, HEMA molecules would cover more of the protein surface. Consequently, in the latter case, many potentially autoreactive B-cells would be excluded and substituted with HEMA-binding B-cells.

It may also be speculated that autoantibody activity toward native albumin could be a result of cross-reactivity, with formation of antibodies able to bind to albumin epitopes regardless of the presence of HEMA. This view is not unreasonable when one considers that a single HEMA molecule would occupy only a small portion of an epitope, since protein antigens are considered to make contact with the entire antibody-combining site, comprised of 1400–2300 Ų (Li et al., 2003). This reasoning also provides an explanation for the observed difference in antibody avidity toward native MSA, which was dependent on the conjugation degree that was used at immunization. Antibodies induced to albumin carrying 15 HEMA molecules would probably experience a substantial mismatch on binding to native instead of to HEMA-modified albumin. Hence, they would display a lower avidity to native MSA than would antibodies induced by MSA carrying 3 HEMA molecules, as supported by our findings.

The response to HEMA-modified protein was correlated to the preparation used at immunization. Thus, antibodies induced by MSA_H3 had significantly higher activity toward this antigen preparation than did antibodies induced by other conjugation degrees, while it was significantly lower to MSA_H22. This supports our analysis that, although each preparation probably contains a mixture of conjugates, the majority will be around the average conjugation degree. Behind the observed responses is, of course, a mixture of antibodies with different specificities and affinities, which will mature into higher affinity by mechanisms such as somatic hypermutation and competition for antigen during the course of the immune response (Ziegner et al., 1994; Wang et al., 2000).

A most noteworthy finding in our study was the appearance of IgE autoantibodies in animals immunized with MSA_H3. IgE antibodies cross-reactive with self-protein were also found in a study where mice were immunized with a foreign protein mimicking a self-protein (Bunder et al., 2004). In patients with atopic dermatitis, recent studies have pointed at a correlation between disease severity and the levels of IgE autoantibodies (Natter et al., 1998; Mittermann et al., 2004).

In conclusion, this study has shown that a low level of conjugation of HEMA to a self-protein was sufficient not only to induce, but also to lead to the highest production of autoantibodies to the carrier. Studies are needed to elucidate the potential of HEMA to induce similar autoantibodies in humans.

	ACKNOWLEDGMENTS

 
Grants from the Swedish Dental Society, the Swedish Research Council (nr K2001-06X-14054-01A), the Swedish Asthma and Allergy Association, and the Faculty of Odontology (TUA), Göteborg, Sweden, are gratefully acknowledged.

Received for publication May 13, 2004. Revision received March 1, 2005. Accepted for publication March 2, 2005.

	REFERENCES

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Journal of Dental Research, Vol. 84, No. 6, 537-541 (2005)
DOI: 10.1177/154405910508400610


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