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J Dent Res 83(1): 60-64, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Identification of in vivo Pellicle Constituents by Analysis of Serum Immune Responses

J. Li1, E.J. Helmerhorst1, R.F. Troxler1,2, and F.G. Oppenheim1,*,2

1 Department of Periodontology and Oral Biology, Boston University Goldman School of Dental Medicine, 700 Albany Street, Boston, MA 02118, USA; and
2 Department of Biochemistry, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA;

* corresponding author, fropp{at}bu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human acquired enamel pellicle is composed of molecules that selectively adsorb from saliva onto tooth surfaces and provides a protective interface between the tooth enamel and the oral environment. To identify the micro-amounts of components present in pellicle, we immunized mice with in vivo-formed human acquired enamel pellicle and analyzed the serum immune responses. Selective reactivities of the serum (OD > 1.0 above background) against albumin, amylase, carbonic anhydrase II, sIgA, IgG, IgM, lactoferrin, lysozyme, proline-rich proteins, statherin, histatin 1, and mucous glycoprotein 1 were observed. We further confirmed the presence of proline-rich proteins, lactoferrin, lysozyme, and carbonic anhydrase II by probing in vivo pellicle with specific polyclonal anti-sera. The polyclonal antibody approach provided a powerful method for the identification of various pellicle proteins, including some which show mineral homeostasis or antimicrobial activity.

KEY WORDS: in vivo pellicle • serum • immune response • composition • proteins


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human acquired enamel pellicle is formed by proteins, peptides, and other organic molecules which selectively adsorb onto tooth surfaces. Pellicle exhibits several biological functions, such as the formation of a protective interface between the tooth surface and the oral environment (Levine et al., 1985), acting as a selective permeability barrier which regulates mineralization/demineralization processes (Moreno and Zahradnik, 1979) and dictating the composition of the microbial biofilm that forms on the tooth surface (Gibbons, 1989). We must know the composition of pellicle if we are to understand the molecular principles that mediate these functional processes and offer the opportunity to interfere with these processes. In vitro studies have shown that some salivary proteins—such as albumin, amylase, cystatin, histatins, IgA, IgG, lactoferrin, lactoperoxidase, lysozyme, proline-rich proteins (PRPs), and statherin—exhibit high affinity for hydroxyapatite powder or enamel slab surfaces (Pruitt and Adamson, 1977; Rølla et al., 1983; Jensen et al., 1992; Hirano et al., 2000). Whether these proteins are constituents of pellicle formed in vivo, however, remains to be elucidated.

A major obstacle in in vivo pellicle studies is the small amounts of proteins that can be harvested from the tooth surfaces. A novel approach to the investigation of pellicle composition is analysis of the immune response in mice elicited by human in vivo-formed pellicle. Multiple encounters of the immune system with a certain antigen amplify the response with great accuracy, which enables only trace amounts of pellicle proteins harvested from tooth surfaces to be detected. In our previous study, we demonstrated that monoclonal antibodies can be generated against mucous glycoprotein 1 (MG1), albumin, amylase, Immunoglobulins (Igs), statherin, and histatin 1 from mice immunized with pellicle, indicating that these proteins, or portions of these proteins, are pellicle constituents. On the other hand, no monoclonal antibody was found against PRPs, lysozyme, lactoferrin, or carbonic anhydrase (Li et al., 2003). The absence of monoclonal antibodies against PRPs, lysozyme, and lactoferrin was surprising in view of the fact that these proteins do possess a high affinity for hydroxyapatite in vitro (Rølla et al., 1983; Jensen et al., 1992; Hirano et al., 2000). Therefore, in the current study, we analyzed the activity of the serum of pellicle-immunized mice instead of using their spleens for monoclonal antibody production. This approach provides a direct assessment of the immunological response generated by human acquired enamel pellicle, which could be related to its composition.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Proteins and Antibodies
Purified MG1 (Troxler et al., 1995) and MG2 (Liu et al., 1999), acidic proline-rich proteins (PRPs) (Oppenheim et al., 1971), histatins 1, 3, 5 (Oppenheim et al., 1988), and statherin (Jensen et al., 1991) were isolated from salivary secretions as described. Albumin (from human plasma), {alpha}-amylase (from human saliva), lysozyme (from human neutrophils), lactoferrin (from human milk), lactoperoxidase (from bovine milk), carbonic anhydrase II (from human erythrocytes), IgG, IgM, and complement factor 1 (from human serum), fibronectin (from human plasma), insulin B chain (from bovine pancreas), cystatin (from chicken egg white), and collagen (from calf serum) were purchased from Sigma (St. Louis, MO, USA). Purified human sIgA (from colostrum) was purchased from Accurate Chemical & Scientific Corporation (Westbury, NY, USA). Human recombinant epithelial growth factor (EGF) was purchased from ICN biochemicals (Aurora, OH, USA).

Rabbit anti-human lysozyme and rabbit anti-human lactoferrin anti-sera were purchased from ICN Biochemicals (Aurora, OH, USA). Goat anti-PRP1 antibodies were prepared by Lofstrand Labs (Gaithersburg, MD, USA). This antiserum showed immune reactivity toward all acidic PRPs, due to sequence homologies within this protein family, but did not cross-react with other salivary proteins (unpublished observations). Rabbit anti-human carbonic anhydrase II and anti-human carbonic anhydrase VI anti-sera were generous gifts from Dr. S. Parkkila, Institute of Medical Technology, University of Tampere, Finland. The preparations and specificities of both antisera have been described in previous reports (Parkkila et al., 1990, 1994). Horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG, HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated rabbit anti-mouse IgG were purchased from Pierce (Rockford, IL, USA).

In vivo Pellicle Collection
The protocol for this investigation was approved by the Institutional Review Board of Boston University Medical Center, and informed consent was obtained from all subjects. Human pellicle formed in vivo for 2 hrs was harvested from intact tooth surfaces by a mechanical-chemical dissociation method involving PVDF membrane (Durapore, 45-µm pore size; Millipore, Bedford, MA, USA) soaked in 0.5 M sodium bicarbonate buffer, pH 8.4, as described (Yao et al., 2001). For pellicle collection, we isolated teeth with cotton rolls after pumicing and rinsed them twice with water to avoid saliva contamination. Pellicle proteins were dissociated from the membranes by being vortexed for 30 sec followed by sonication for 5 min in water. The protein content in the eluate was determined with a bi-cinchoninic acid (BCA) protein assay (Micro BCA Protein Assay Reagent Kit; Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Aliquots of 50 µg were evaporated to dryness by means of a Vacufuge Concentrator (Brinkmann Instruments, Westbury, NY, USA) and stored at -20°C.

Pellicle samples used in gel electrophoresis were desalted by sequential dilution-centrifugation steps in an Amicon microcentrifuge device (Millipore, Bedford, MA, USA) with a molecular-weight cut-off of 3 kDa. The protein content in the dialysate after the desalting process was determined by the BCA method.

Immunological Procedures
Lyophilized pellicle samples (50 µg) were reconstituted in 100 µL PBS solution, and mixed vigorously with an equal volume (100 µL) of adjuvant (RIBI Adjuvant System, RIBI ImmunoChem Research, Inc., Hamilton, MT, USA) for 2–3 min until a thick emulsion developed. The resulting 200-µL quantity of immunogen emulsion was injected intraperitoneally to induce a humoral immune response in 2 six-week-old female BALB/c mice (Charles River Laboratories, Boston, MA, USA), according to the immunization procedure described by Harlow and Lane (1988). Four subsequent "boosts", one month apart, were carried out in exactly the same way. The protocol for this investigation was approved by the Institutional Animal Care and Use Committee of Boston University Medical Center. We processed pre-immune blood, as well as blood collected ten days after the fourth boost, to obtain the serum as described by Harlow and Lane (1988).

Immunological Assays
    Enzyme-linked immunosorbent assay (ELISA)
The 96-well ELISA microtiter plates (Greiner, Frederick, MD, USA) were coated with 50 µL of pellicle protein mixture (10 µg/mL) or one of the selected salivary proteins (2 µg/mL). Plates were washed and incubated with a 1:1000 dilution of mice pre- or post-immune serum in TBST (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20, pH 7.5) for 2 hrs at room temperature. Bound antibodies were detected with a 1:2500 dilution of HRP-conjugated rabbit anti-mouse IgG antibody (Pierce), with OPD (o-phenylenediamine dihydrochloride; Pierce) as the substrate. Reactions were analyzed spectrophotometrically at 490 nm.

    Polyacrylamide gel electrophoresis (PAGE) and Western blotting
In vivo pellicle samples and purified protein standards were subjected to either Tris-tricine (Ausubel et al., 1995) or Ornstein-Davis (Davis, 1964; Ornstein, 1964) PAGE, with 10% and 15% acrylamide concentrations in the separating gels, respectively, blotted onto PVDF membranes and incubated with the appropriate specific antibody for 1 hr at room temperature. Bound Abs were detected with HRP-conjugated secondary Abs (rabbit anti-goat IgG or goat anti-rabbit IgG; Pierce), with chloronaphthol/diaminobenzidine (CN/DAB kit; Pierce) as the substrate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
To assess the composition of pellicle, we immunized 2 mice with in vivo-formed human acquired enamel pellicle. The reactivities of the pre- and post-immune antisera against several salivary proteins were tested by ELISA. Pre-immune serum of both mice showed negligible reactivity toward pellicle or any of the selected salivary proteins. Post-immune serum from both mice showed nearly identical and selective reactivities toward certain salivary proteins (Fig. 1Go). Strong reactivity (OD > 2.0) was observed toward pellicle, albumin, amylase, carbonic anhydrase II, sIgA, IgG, lactoferrin, lysozyme, PRPs, and statherin; medium reactivity (1.0 < OD < 2.0) was observed toward histatin 1, IgM, and MG1; and low or negligible reactivity (OD < 1.0) was observed toward complement factor 1, cystatin, EGF, fibronectin, histatin 3, histatin 5, insulin B, lactoperoxidase, and MG2. The binding specificity of the post-immune serum was further demonstrated by the fact that no reactivity was observed toward collagen, a non-salivary protein (Fig. 1Go, Panels A and B). These results indicate that a specific and reproducible serum immune response against several salivary proteins can be obtained upon immunization of mice with human pellicle, strongly suggesting that these salivary proteins, or portions thereof, are pellicle constituents.



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  		Figure 1. Reactivities of mouse anti-pellicle antiserum against purified protein standards in ELISA. Pre- and post-immune sera were collected from 2 mice before the first injection with pellicle and ten days after the 4th boost with pellicle proteins, respectively. Microtiter plates were coated with pellicle proteins (10 µg/mL) or a series of purified protein standards (2 µg/mL) and subsequently incubated with 1:1000 diluted pre-immune serum (white bars) or 1:1000 diluted post-immune serum (black bars). Each bar represents the mean value ± SD of one experiment performed in triplicate. Panel A, mouse #1; Panel B, mouse #2.

 
Reactivities of post-immune anti-sera with MG1, albumin, amylase, immunoglobulins, statherin, and histatin 1 were consistent with results of our previous study, in which monoclonal antibodies were obtained against these proteins, confirming that these proteins are in vivo pellicle constituents (Li et al., 2003). In addition to these proteins, the post-immune serum showed high binding activity toward PRPs, lysozyme, lactoferrin, and carbonic anhydrase II, indicating that these proteins might be in vivo pellicle constituents as well. To exclude that a particular reactivity was due to cross-reactivity of polyclonal antiserum with overlapping or similar epitopes in different proteins, we used a series of specific anti-sera to test the presence of the positively reacting proteins in pellicle by Western blotting. The detection of an immunoreactive band with mobility identical to that of the purified protein standard demonstrates the presence of the corresponding protein in in vivo pellicle. Immunoreactive bands could be detected in in vivo pellicle with mobility similar to that of purified lysozyme, lactoferrin, carbonic anhydrase II, and PRP1 (Fig. 2Go, Panels A-D). The anti-PRP anti-serum, which recognizes both large and small acidic PRPs (Schüpbach et al., 2001), recognized multiple bands in pellicle (Fig. 2Go, Panel D), consistent with the fact that multiple family members of acidic PRPs were recognized by the polyclonal anti-pellicle antisera from mice immunized with human pellicle (Fig. 1Go).



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  		Figure 2. Immunoblotting of human pellicle, purified lysozyme, lactoferrin, carbonic anhydrase II, and PRP1 with specific antisera. Purified proteins (0.25 µg) or pellicle (40 µg) was subjected to Tris-tricine (Panels A-C) or Ornstein-Davis (Panel D) PAGE and blotted onto PVDF membranes. Panel A, blot was probed with 1:100 diluted anti-lysozyme anti-serum. Panel B, blot was probed with 1:250 diluted anti-lactoferrin anti-serum. Panel C, blots were probed with 1:1000 diluted anti-carbonic anhydrase II anti-serum. Panel D, blot was probed with 1:2000 diluted anti-PRP1 anti-serum. Upper arrow, large PRPs (PRP1, 2 and PIFs); lower arrow, small PRPs (PRP3, 4 and PIFf).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we investigated the mice humoral immune response toward human acquired enamel pellicle as a means to elucidate its composition. Strong immunoreactivity was observed toward albumin, amylase, carbonic anhydrase II, sIgA, IgG, lactoferrin, lysozyme, PRPs, and statherin. Medium binding activity was found toward histatin 1, IgM, and MG1. No or low binding activity was found against complement factor 1, cystatin, EGF, fibronectin, histatin 3, histatin 5, insulin B, lactoperoxidase, and MG2. The data were, in part, consistent with our previous study, where we used monoclonal antibody preparation following immunization with in vivo pellicle. Some components—like PPRs, lysozyme, lactoferrin, and carbonic anhydrase II—that were recognized by the anti-pellicle antiserum did not generate monoclonal antibodies in our previous study (Li et al., 2003). One possible explanation for this finding is that, during the monoclonal antibody preparation procedure, a specific antibody-secreting B-cell is not selected during the fusion procedure with a myeloma cell, leading to a false-negative result. Another observation was that some components toward which monoclonal antibodies were obtained in our previous study, such as MG1 and histatin 1, showed only moderate serum immune responses. It should be noted that the secondary antibody that was used in the present study was directed against IgG, since IgG isotype antibodies are more mature and less likely to display cross-reactivity than are IgM isotype antibodies. It is likely that the anti-histatin 1 and anti-MG1 responses were of the IgM isotype and therefore showed little reactivity with the anti-IgG antibodies (Li et al., 2003).

Some components—such as complement factor 1, cystatin, EGF, fibronectin, histatin 3, histatin 5, insulin B, lactoperoxidase, and MG2—did not appear to generate monoclonal antibodies or to yield medium or strong polyclonal antibody responses in anti-pellicle antisera. It is important to note that the absence of an immune response against a certain antigen does not necessarily mean the absence of that component in pellicle, since the response is dependent not only on the abundance but also on the immunogeneity and the presentation of a certain component to the immune system.

The identification of acidic PRPs, statherin, and MG1 in human in vivo pellicle is of biological importance, since acidic PRPs, statherin, and MG1 can provide binding sites for certain bacteria after being adsorbed onto hydroxyapatite (Gibbons and Hay, 1988, 1989; Gibbons et al., 1988; Lamblin et al., 1992; Nieuw Amerongen et al., 1995). This indicates that these proteins, when attached to the tooth surface, might function in recruiting micro-organisms to pellicle. At the same time, antimicrobial non-specific host defense factors such as lysozyme, lactoferrin, and histatins were also identified as pellicle constituents. The latter finding indicates that various antimicrobial molecules are recruited to sites where microbial attachment and growth occur on dental surfaces. The simultaneous presence of bacterial adhesion factors and bacterial inhibition factors indicates that pellicle functions in recruiting micro-organisms and at the same time controls their growth, e.g., by direct killing or iron deprivation. It is the cumulative effect from all pellicle constituents that will dictate the final microbial profiles on pellicle.

In this study, two forms of carbonic anhydrase were also found to be part of pellicle. These enzymes are zinc-containing metalloproteins that participate in a variety of physiological processes, such as pH regulation, carbon dioxide and bicarbonate transport, and water and electrolyte balance (Parkkila and Parkkila, 1996). Carbonic anhydrase II is a widely distributed isoenzyme in oral stratified squamous epithelial cells, while carbonic anhydrase VI has been identified as a salivary component (Parkkila et al., 1990, 1994). Carbonic anhydrase VI has been previously located in human in vivo pellicle on extracted teeth (Leinonen et al., 1999). Our results confirmed that salivary carbonic anhydrase VI is indeed an in vivo pellicle constituent (data not shown) and indicated that, in addition to salivary carbonic anhydrase VI, carbonic anhydrase II is also present (Fig. 2Go, Panel C). Previous studies have shown that carbonic anhydrase II deficiency can directly or indirectly affect the expression of the salivary carbonic anhydrase VI (Murakami and Sly, 1987). It could be that these two isoenzymes also function cooperatively. The presence of both carbonic anhydrases II and VI in in vivo pellicle suggests that they might represent an important and mutually complementary buffer capacity regulatory system to regulate plaque pH by accelerating acid removal in the local environment of the tooth surface, thereby preventing caries formation.

In conclusion, analysis of mice serum immune reactivity induced by human pellicle, followed by confirmation of the presence of the positively reacting proteins with specific antisera, represents a complementary approach to monoclonal antibody preparation. These immunological methodologies can be successfully used for the elucidation of the composition of human acquired enamel pellicle.


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Dr. Yuan Yao for providing purified protein standards and Dr. Seppo Parkkila for providing anti-human carbonic anhydrase II and anti-human carbonic anhydrase VI antisera. This study is supported by NIH/NIDCR grants DE 05672, DE 07652, DE 11691, and DE 14950.

Received May 12, 2003; Last revision October 10, 2003; Accepted October 16, 2003


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