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Biomaterials & Bioengineering |
HEMA Bound to Self-protein Promotes Auto-antibody Production in Mice
E. Sandberg1,*,
G. Bergenholtz1,
C. Eklund1 and
U.I. Dahlgren1,2
1 Department of Endodontology/Oral Diagnosis, Faculty of Odontology, Box 450, SE 405 30 Göteborg; and
2 Department of Rheumatology, Faculty of Medicine, The Sahlgrenska Academy at Göteborg University, Sweden;
Correspondence: * corresponding author, Elisabeth.Sandberg{at}odontologi.gu.se
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ABSTRACT
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While several studies report that acrylic monomers contained in dental materials may cause hypersensitivity reactions, little is known of the associated immune response. Here we address the potential of 2-hydroxyethyl-methacrylate (HEMA) to bind to endogenous protein and elicit auto-antibody production in vivo. Albumin was incubated with HEMA at various times and pH. Following confirmation of the conjugation by inhibition of trinitrophenyl (TNP) binding, female Balb/c mice received HEMA conjugated to mouse serum albumin (MSA) in Freund’s incomplete adjuvant or saline subcutaneously. ELISA was used to determine the serum antibody responses to native and modified MSA. IL-2 production in spleen cell cultures stimulated with HEMA-conjugated MSA was measured. HEMA reacted with serum albumin at physiological conditions. HEMA-conjugated MSA induced IL-2 secretion and production of IgG antibodies to native MSA. The results suggest that modification of an endogenous protein like serum albumin with HEMA may defeat the control of immune responses to this self-protein.
Key Words: acrylates • autoimmunity • biomaterials • dental material
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INTRODUCTION
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Several reports have made plausible claims that constituents in resin composites may produce adverse biological effects, including allergic reactions (Björkner, 1981; Murer et al., 1995; Gebhart and Geier, 1996; Kanerva et al., 1997, 2000; Bong and English, 2000; Geukens and Goossens, 2001). For example, in a German study of 55 dental technicians with occupational skin disease, 64% were found to have allergic contact dermatitis (Rustemeyer and Frosch, 1996). Of these individuals, 33% were sensitive to 2-hydroxyethylmethacrylate (HEMA). While reports on similar responses in patients with composite restorations are lacking, experiments have shown that guinea pigs subjected to acrylates develop responses resembling contact hypersensitivity reactions (Katsuno et al., 1996). Attempts to develop a murine model for the study of acrylate hypersensitivity have been unsuccessful (Rustemeyer et al., 1998).
An immune response to low-molecular-weight substances requires binding to a protein carrier, which in vivo is likely to be of self-origin. Albumin is an abundant, both intra- and extravasculary, protein (Peters, 1996). It contains 59 lysine residues that are likely to react with the double bond of HEMA, thereby creating a covalent bond (Roberts, 1987; Peters, 1996). Here we test the hypothesis that HEMA can bind to mouse serum albumin (MSA) and induce an immune response upon challenge in mice to a breach in B-cell self-tolerance with a subsequent production of anti-albumin antibodies.
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MATERIALS & METHODS
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Conjugation of HEMA to Protein
HEMA (H8633; Sigma Chemical Co., St. Louis, MO, USA) was incubated with bovine serum albumin (BSA) (Sigma, Fraction V, A4503) at pH 7.4 in 0.15 M phosphate-buffered saline (PBS) or at pH 8.1 in 0.1 M HCO3 buffer. Incubations were performed at a constant molecular ratio of 500:1 of HEMA to protein. At various times, reaction was stopped by diafiltration on a filter device with a molecular cutoff of 30 kD (Nanosep® Centrifugal devices OD030C33, Pall Gelman Sciences, Lund, Sweden).
Titration of Free Lysine Residues
2,4,6-Trinitrobenzenesulfonic acid (TNBS) readily reacts with the lysine residues of a protein; the sulphonic group is cleaved off, leaving trinitrophenyl (TNP) covalently bound to the nitrogen (Sashidhar et al., 1994). The number of HEMA monomers bound to protein (conjugation degree) was obtained by analysis of TNP conjugation with or without prior HEMA treatment.
A 100-µg quantity of protein in 1 mL PBS buffer, 1 mL 4% NaHCO3, and 1 mL 0.01% TNBS solution (Sigma, P2297) was incubated for 2 hrs at 42°C. The reaction was stopped with 1 mL 10% sodium dodecyl sulphate and 0.5 mL 1 M HCl. Absorbance was read at 335 nm (Sashidhar et al., 1994).
Preparation of H-MSA
Mouse serum albumin (MSA, 13.2 mg) (Sigma, fraction V, A3139) was incubated in 0.1 M NaHCO3 buffer mixed with 23 µL HEMA for 67 hrs at 37°C. The mixture was diafiltrated 4x with PBS (Dulbecco’s, Gibco BRL 14190-094, Life Technologies AB, Täby, Sweden). Purity was checked spectrophotometrically. Control MSA incubated without HEMA was treated similarly. The conjugation degree of HEMA to MSA was 23%, i.e., 11 of the calculated 48 lysine residues of MSA were occupied with HEMA (Peters, 1996). The final product was filtered through a sterile 0.2-µm filter (Acrodisc® Syringe Filter, ref. 4454, Pall Gelman) before storage in aliquots at -20°C until use. In some cases, HEMA was passed over a column (Aldrich No 30631-2, Sigma-Aldrich) to remove the inhibitor hydroquinone, resulting in a conjugation degree of 34%.
Immunization
Female 7- to 8-week-old BALB/C mice were immunized subcutaneously with 50 µg H-MSA (conjugation degree 23%) or MSA in 0.05 mL PBS, mixed with an equal volume of Freund’s incomplete adjuvant (FICA). Eight animals were sham-immunized with PBS/FICA. Three similar booster injections were given 3 wks apart. Six days after the last booster, the animals were killed. In some experiments, the adjuvant were excluded, and the animals were immunized with 0.4 mg protein and "boostered" twice with 0.2 mg. Serum was kept frozen until analysis. The Göteborg ethical committee on animal experiments approved the animal protocols.
ELISA
Plates (Nunc immuno plate, MaxisorpTM, Kamstrup, Denmark) were coated overnight in 4°C with MSA or H-MSA 5 µg/mL in PBS buffer (pH 7.3) and washed 3 x with PBS-TWEEN before incubation with serum in appropriate dilutions overnight at 4°C. This was followed by 4 PBS-Tween washes. Biotin- conjugated anti-mouse IgG (Jackson Immuno Research Laboratories, Inc., West Grove, PA, USA), IgG1, IgG2a, IgG2b, or IgG3 antibodies (BD Pharmingen, Heidelberg, Germany) were added for 2 hrs at room temperature. Plates were washed 4x and incubated with extravidin-alkaline phosphatase (Sigma E2636) for 2 hrs, followed by another 4 washes. The substrate paranitrophenyl (104 Sigma) 1 mg/mL, dissolved in diethanolamine buffer, pH 9.8, was added. The 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.
IL-2 Secretion
Spleens were squeezed through a 70-µM cell strainer (Falcon, Labora AB, Sollentuna, Sweden), washed, and centrifuged on Ficoll-PaqueTM (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Cells from the interface were washed 2x and then suspended in Dulbecco’s MEM, supplemented with 5% fetal calf serum, penicillin-streptomycin, and gentamicin (all from Gibco BRLTM, Life Technologies). Cells were seeded 5x105 cells/well (0.2 mL) in triplicate on a 96-well plate and stimulated with MSA 40 µg/well for 4 days. Supernatants were collected and analyzed for IL-2 (Duoset ELISA, nr DY402, RD Systems, Abingdon, UK) according to the manufacturer’s instruction.
Statistics
The Mann-Whitney U-test was used in the statistical analysis.
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RESULTS
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Binding of HEMA to Albumin—Influence of Concentration of Reactants, pH, and Incubation Time
Increasing the concentration of protein led to increased numbers of HEMA molecules bound to BSA (Fig. 1A ). After 45 hrs of incubation at pH 7.4, the average conjugation ratio at a protein concentration of 40 mg/mL was 4 HEMA molecules per BSA molecule.
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Figure 1. Number of 2-hydroxyethylmethacrylate (HEMA) molecules bound to bovine serum albumin (BSA) after incubation at a molecular ratio (HEMA to BSA) of 500:1. The number of conjugated HEMA molecules was calculated from the subsequent inhibition of trinitrophenyl binding to the BSA molecules. (A) Samples were incubated at 37°C, pH 7.4, for 45 hrs at increasing protein concentrations. Means and standard deviations are from 3 samples. (B) Influence of incubation time and pH at a BSA concentration of 40 mg/mL. Means and standard deviations of 2 samples.
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With incubation at different pH, binding of HEMA to BSA was higher at pH 8.1 than that at pH 7.4 (Fig. 1B ). After 65 hrs of incubation, an average of 9 and 30 HEMA molecules was bound to each BSA molecule, respectively.
The number of HEMA molecules conjugated to each BSA molecule increased by time (Fig. 1B). At pH 7.4, the reaction between HEMA and protein seemed to occur at a constant pace, while the reaction proceeded more rapidly during the first 20 hrs of incubation, at pH 8.1.
Antibody Responses
Mice were immunized with H-MSA or native MSA in FICA. Animals immunized with H-MSA produced significantly more auto-antibodies against MSA than mice immunized with native MSA (Fig. 2A). Animals given adjuvant alone produced no anti-MSA antibodies.
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Figure 2. IgG anti-MSA (A) and anti H-MSA (B) antibody activity in serum of mice immunized with mouse serum albumin carrying HEMA (H-MSA) (n = 9), mouse serum albumin (MSA) (n = 9), or adjuvant (n = 8) only. The mice were immunized in Freund’s incomplete adjuvant 4x with intervals of 2 to 3 wks. Serum was taken 6 days after the last immunization. Each dot represents 1 mouse, and the median is indicated. Statistical comparison was done between the MSA- and H-MSA-treated groups. The IgG antibody activity was determined with ELISA and is expressed as absorbance units at 405 nm, serum dilution 1/100.
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Animals immunized with HEMA-modified albumin in FICA developed IgG antibodies against H-MSA (Fig. 2B), which were not possible to absorb with MSA. IgG antibody activity against H-MSA was also found in a few MSA-immunized mice, while animals given adjuvant alone were negative.
IgG1, IgG2a, IgG2b, and IgG3 antibody activity to native MSA was measured and related to the IgG antibody activity. The relative IgG1 anti-MSA antibody response was greater in animals immunized with the HEMA-modified MSA than in the animals immunized with native MSA (Fig. 3). The IgG2a and the IgG2b anti-MSA antibody response was low and did not differ between the groups (not shown). Animals were negative for IgG3 antibodies.
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Figure 3. The IgG1 anti-MSA antibody activity in relation to the corresponding IgG anti-MSA antibody activity in serum of mice immunized with mouse serum albumin carrying HEMA (H-MSA) (n = 9), mouse serum albumin (MSA) (n = 9), or adjuvant (n = 8) only. The mice were immunized 4x 2 to 3 wks apart, and the serum was taken 6 days after the last immunization. Each dot represents 1 mouse, and the median is indicated. Statistical comparison was done between the MSA- and H-MSA-treated groups.
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T-cell Responses
Spleen cells from mice immunized with H-MSA were stimulated in vitro with H-MSA. The cells from the mice immunized with H-MSA produced significantly more IL-2 than the control mice (Fig. 4).
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Figure 4. IL-2 secretion of spleen cell cultures stimulated with HEMA-conjugated mouse serum albumin (H-MSA). Mice were immunized subcutaneously with H-MSA (n = 8) without adjuvant. Controls (n = 8) were given phosphate-buffered saline only. The mice were immunized 3x and killed 7 days after the last immunization. Supernatants were analyzed for IL-2 after 4 days’ stimulation with H-MSA. Each dot represents 1 mouse, and the median is indicated.
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DISCUSSION
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The present study demonstrates that auto-antibodies were produced when mice were immunized with HEMA-modified auto-protein. Obviously, the control of the immune response to this self-protein was broken by the HEMA treatment. This particular aspect of the potential adverse effects of HEMA has not been previously highlighted, while numerous studies have been concerned with a variety of non-specific reactions. For example, in vitro studies have shown that macrophages exposed to polymethylmethacrylate particles secrete interleukin-6 and tumor necrosis factor (Trindade et al., 1999), and that their proliferation is impaired by sub-toxic concentrations of HEMA (Bouillaguet et al., 2000). Exposure to acrylates also lowers T-cell proliferation (Aronsson et al., 2000). Yet, one study on adaptive immune responses found that T-cells primed in vitro with dendritic cells, pre-incubated with HEMA, responded specifically to HEMA with proliferation and interferon-gamma production (Rustemeyer et al., 1999). The results of that study are in line with the findings of the present study.
Immunization with the native self-protein MSA in adjuvant led to a limited production of antibodies to MSA. This is in agreement with the presence of a mechanism which down-regulates or prevents production of antibodies that can react with self-components. HEMA-modified MSA breached this suppression, resulting in a pronounced production of IgG antibodies against native MSA. A possible mechanism to explain this finding could be that autoreactive B-cells, specific for epitopes on MSA, internalized the H-MSA antigen and presented HEMA-modified peptides for specific T-cells. These T-cells would then help B-cells to produce auto-antibodies to MSA. Normally, an immune response to MSA would not be possible, since T-cells specific for self-protein such as native MSA are deleted in the thymus (Sakaguchi et al., 1995). Indeed, the secretion of IL-2 indicates the presence of H-MSA-specific T-cells in the animals immunized with H-MSA that could help auto-reactive B-cells.
The capacity of HEMA-modified MSA to endorse auto-antibody production was even more evident when the subclass profile of the antibody response was analyzed. The animals immunized with the modified self-protein had a much higher relative production of IgG1 anti-MSA antibodies than did the animals immunized with the native MSA. This pattern of response is congruent with a Th2 type of immune response (Yan et al., 2000).
The generation of auto-antibodies is not equal to the appearance of autoimmune disease. While such antibodies constitute a potential hazard, clinical manifestations of HEMA-induced immunity to self remain to be demonstrated. One may argue, given the fact that binding of HEMA to BSA was lower at physiologic pH than at pH 8.1, the risk would be low under in vivo conditions. Since acrylate binding increased over time, long-term exposure to acrylates may nevertheless result in substantial protein conjugations. Also, pH can vary locally and be above 7.4 in the oral cavity, for example, which would facilitate conjugation. Therefore, when potential adverse biological effects of modern dental materials are assessed, their potential to bind to protein should be taken into consideration.
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ACKNOWLEDGMENTS
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Grants from The Swedish Research Council (no. K2001-06X-14054-01A), the Swedish Asthma and Allergy Association, the Wilhelm and Martina Lundgren Foundation, and the Faculty of Odontology (TUA), Göteborg, Sweden, are gratefully acknowledged.
Received for publication December 6, 2001.
Revision received June 28, 2002.
Accepted for publication July 3, 2002.
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REFERENCES
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- Aronsson G, Dahlgren UI, Karlsson S (2000). Human and rat mononuclear cell proliferation show different sensitivity, in vitro, to single constituents of dental composite resins. J Biomed Mater Res 53:651–657.[Medline]
[Order article via Infotrieve]
- Björkner B (1981). Sensitization capacity of acrylated prepolymers in ultraviolet curing inks tested in the guinea pig. Acta Derm Venereol 61:7–10.[Medline]
[Order article via Infotrieve]
- Bong JL, English JS (2000). Allergic contact dermatitis from airborne exposure to acrylates. Contact Dermatitis 43:242.[Medline]
[Order article via Infotrieve]
- Bouillaguet S, Wataha JC, Virgillito M, Gonzalez L, Rakich DR, Meyer JM (2000). Effect of sub-lethal concentrations of HEMA (2-hydroxyethyl methacrylate) on THP-1 human monocyte-macrophages, in vitro. Dent Mater 16:213–217.[CrossRef][Medline]
[Order article via Infotrieve]
- Gebhart M, Geier J (1996). Evaluation of patch test results with denture material series. Contact Dermatitis 34:191–195.[Medline]
[Order article via Infotrieve]
- Geukens S, Goossens A (2001). Occupational contact allergy to (meth)acrylates. Contact Dermatitis 44:153–159.[Medline]
[Order article via Infotrieve]
- Kanerva L, Jolanki R, Estlander T (1997). 10 years of patch testing with the (meth)acrylate series. Contact Dermatitis 37:255–258.[CrossRef][Medline]
[Order article via Infotrieve]
- Kanerva L, Alanko K, Estlander T, Jolanki R, Lahtinen A, Savela A (2000). Statistics on occupational contact dermatitis from (meth)acrylates in dental personnel. Contact Dermatitis 42:175–176.[Medline]
[Order article via Infotrieve]
- Katsuno K, Manabe A, Itoh K, Nakamura Y, Wakumoto S, Hisamitsu H, et al. (1996). Contact dermatitis caused by 2-HEMA and GM dentin primer solutions applied to guinea pigs and humans. Dent Mater J 15:22–30.[Medline]
[Order article via Infotrieve]
- Murer AJ, Poulsen OM, Roed-Petersen J, Tuchsen F (1995). Skin problems among Danish dental technicians. A cross-sectional study. Contact Dermatitis 33:42–47.[Medline]
[Order article via Infotrieve]
- Peters T Jr (1996). All about albumin: biochemistry, genetics and medical applications. New York: Academic Press Inc.
- Roberts DW (1987). Structure-activity relationships for skin sensitisation potential of diacrylates and dimethacrylates. Contact Dermatitis 17:281–289.[CrossRef][Medline]
[Order article via Infotrieve]
- Rustemeyer T, Frosch PJ (1996). Occupational skin diseases in dental laboratory technicians. (I) Clinical picture and causative factors. Contact Dermatitis 34:125–133.[Medline]
[Order article via Infotrieve]
- Rustemeyer T, de Groot J, von Blomberg BM, Frosch PJ, Scheper RJ (1998). Cross-reactivity patterns of contact-sensitizing methacrylates. Toxicol Appl Pharmacol 148:83–90.[Medline]
[Order article via Infotrieve]
- Rustemeyer T, De Ligter S, Von Blomberg BM, Frosch PJ, Scheper RJ (1999). Human T lymphocyte priming in vitro by haptenated autologous dendritic cells. Clin Exp Immunol 117:209–216.[Medline]
[Order article via Infotrieve]
- Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M (1995). Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol 155:1151–1164.[Abstract]
- Sashidhar RB, Capoor AK, Ramana D (1994). Quantitation of epsilon-amino group using amino acids as reference standards by trinitrobenzene sulfonic acid. A simple spectrophotometric method for the estimation of hapten to carrier protein ratio. J Immunol Meth 167:121–127.[CrossRef][Medline]
[Order article via Infotrieve]
- Trindade MC, Lind M, Goodman SB, Maloney WJ, Schurman DJ, Smith RL (1999). Interferon-gamma exacerbates polymethylmethacrylate particle-induced interleukin-6 release by human monocyte/macrophages in vitro. J Biomed Mater Res 47:1–7.[Medline]
[Order article via Infotrieve]
- Yan XM, Guo J, Pichurin P, Tanaka K, Jaume JC, Rapoport B, et al. (2000). Cytokines, IgG subclasses and costimulation in a mouse model of thyroid autoimmunity induced by injection of fibroblasts co-expressing MHC class II and thyroid autoantigens. Clin Exp Immunol 122:170–179.[CrossRef][Medline]
[Order article via Infotrieve]
Journal of Dental Research, Vol. 81, No. 9,
633-636 (2002)
DOI: 10.1177/154405910208100911
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ABSTRACT
INTRODUCTION