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Clin Exp Immunol. Feb 2005; 139(2): 328–337.
PMCID: PMC1809282

Identification of epithelial auto-antigens associated with periodontal disease


We previously reported evidence that patients with periodontitis have serum antibodies to oral Gram positive bacteria that are cross-reactive with epithelial antigens. In the present report cross-reactive epithelial antigens including CD24, lactate dehydrogenase A [LDM-A], antioxidant protein 2 [AOP 2] and nuclear factor of activated T cells 5 [NFAT 5], were identified by screening a cDNA expression library with pooled patient sera. Titres of antibodies to CD24 peptide correlated negatively with indices of periodontal disease severity. Strong expression of CD24 in the reactive periodontal epithelium and inflamed gingival attachment contrasted with low to undetectable expression in the external gingival epithelium. In periodontitis, a local action of these auto-reactive antibodies could modulate the regulatory potential associated with expression of CD24 in this epithelium.

Keywords: CD24, autoimmunity, reactive epithelium, periodontitis


Persistent stimulation by antigens of the complex microbial biofilm that colonizes the gingival sulcus results in a failure of the epithelial attachment to the tooth and the apical migration of the reactive epithelium in some sites to form a cleft or pocket that is characteristic of the lesion of periodontitis. Perturbation of the structure of this epithelial lining is a feature of the immunopathological response to bacterial antigens considered to be central in the pathogenesis of the destructive lesion of periodontitis [1]. Serum antibodies reactive with self-antigens including desmosomal proteins [2] and type I collagen [3] have been reported to be increased in patients with chronic periodontitis. One mechanism for the generation of these antibodies is the existence of shared antigenic determinants in exogenous agents such as the oral microflora. In a previous report [4], a relationship between Gram positive bacteria adjacent to diseased sites and the presence of epithelial-reactive IgG antibodies was established. The nature of the epithelial antigens recognized by cross-reactive serum antibodies was further investigated in the present study.

cDNA expression libraries displayed in Lambda phage have been successfully employed to identify partners involved in antibody-antigen, protein-protein and DNA–protein interactions and represent an informative approach to functional genomics. CD24 was one of six products detected by screening an epithelial cDNA expression library. This antigen was selected for further study in relation to the potential importance in regulation of both epithelial biology and the immune response together with the availability of essential reagents.

The processed form of CD24 comprises a peptide core of some 31–35 amino acids linked through a glycosyl phosphatidylinositol anchor to the plasma membrane of expressing cells [5]. Abundant threonine and serine residues serve as glycosylation sites and related to tissue source, the glycosylated forms have molecular weights of 35–60 kD [6]. CD24 is a homologue of murine nectadrin, also known as heat stable antigen, a cell surface glycoprotein implicated in cell-cell adhesion and signalling [7]. It is not considered to be a consistent marker of stratified squamous epithelia at maturity although this antigen may be transiently expressed related to differentiation [8,9]. CD24 is highly expressed in many developing tissues [7,10,11] and in some carcinomas [1215]. The propensity of some neoplasms for haematogenous metastasis has been correlated with high expression of CD24 implicated in mediating invasiveness by ligation of receptors on vascular endothelium [6,12,16]. For instance, heavily glycosylated CD24 may act as a receptor for lectin-like molecules [5], such as P-selectin [17].

In this context, the distribution of CD24 in gingival biopsies was studied with the finding that strong expression was localized to the reactive epithelial components associated with the disease process. It was further determined that serum antibodies reactive with CD24 peptide were associated with an improved diagnosis, suggesting a protective action.

Materials and methods

Clinical data, serum and tissue samples

Blood samples were obtained with informed consent and prior to commencement of therapy from 22 adult chronic periodontitis patients (14 males, 8 females; average age 48 years, range 35–68 years), including 14 with generalized and 8 with localized periodontitis; 9 of 22 patients were tobacco smokers. None of the patients had a history of systemic disease or of antibiotic medication within the preceding 6 months; also no record of periodontal therapy including subgingival scaling, root planing or relevant surgery, in the previous three years. Participants were examined clinically and radiographically then assigned a diagnosis score (1–6) which is a measure of overall status for periodontal disease, as follows: score of 0 = none, 1 = mild local, 2 = mild general, 3 = moderate local, 4 = moderate general, 5 = severe local, 6 = severe general. Six sites per tooth were examined for probing depth and attachment level. Loss of attachment (ranging from a maximum of 5–11 mm for the cohort) which is a measure of long-standing disease activity and formation of periodontal pockets which is a measure of current disease status with maximum probing depths of 4–11 mm for the cohort and resorption of alveolar bone (determined by examination of radiographs), were also recorded.

Blood samples were also collected from 12 ethnically, age, and gender matched non smoking subjects (7 males, 5 females, 27–64 years of age, average 46 years) with clinically healthy gingiva (CHG) characterized by no obvious clinical signs of gingival inflammation, probing depths of 3 mm or less, no sites with significant attachment loss, no radiographic evidence of bone loss. These subjects had no history of systemic disease.

Sera were separated from clotted blood by centrifugation at 1000 g for 20 min and stored at – 70°C until used.

The tissue samples obtained from the same group of 22 periodontitis patients were identified as periodontitis (Perio) sites and minimally inflamed gingival (Min) tissue sites on the basis of clinical record, radiographs and histological examination. Minimally inflamed gingiva (Min) was defined by absence of bleeding on gentle probing, no clinical signs of inflammation, a probing depth of 3 mm or less and histological evidence of only a small number of chronic inflammatory cells in the subepithelial connective tissues. All biopsies yielded serial sections that were satisfactory in relation to tissue preservation and orientation.

Gingival tissues were snap-frozen in isopentane cooled in liquid nitrogen and sections cut at 6 µm. Slides with sequential sections were stored in sealed boxes at −70°C until required.

Library immunoscreening using sera as probes

To define antigens recognized by sera from patients with periodontal disease, a human keratinocyte cDNA library displaying keratinocyte peptides/proteins, containing inserts of size range 0·6–3·0 kb (cat: HL1110b, Clontech Laboratories, Inc., USA) was purchased. This library used the cloning vector λgt11 and an EcoRI cloning site (Lambda library user manual, protocol [sharp] PT1010-1, version [sharp] PR92374, Clontech). The library was screened with pooled IgG from the sera of 22 patients with periodontitis, according to the manufacturer's instructions. Briefly, 0·1 ml diluted library in 1 × lambda dilution buffer (1: 250 000) containing 104 pfu of λgt11 phage, was combined with 0·2 ml E. coli (Y1090r) host strain (OD600≈ 2·0) in LB broth (containing 10 m m MgSO4 and 0·2% maltose) at 37°C for 15 min; mixed with 3 ml LB soft top agarose (containing 10 m m MgSO4) and poured onto 90 mm LB agar (containing 10 m m MgSO4) plates, then incubated at 42°C for 3·5 h to allow growth of lytic phage. IPTG (Isopropyl β-D-thiogalactopyranoside) saturated nitrocellulose filters were placed over the agarose and the plates incubated at 37°C for 3·5 h until growth was nearly confluent. The filters were removed, rinsed in 0·05% Tween 20/TBS (50 m m Tris-buffered saline pH 7·4) and blocked in 20% foetal calf serum (FCS) with 0·05% Tween 20/TBS overnight. Filters were incubated for 1 h at room temperature with pooled sera previously absorbed with E. coli strain Y1090r lysates (see below) until depleted of reactivity for E. coli antigens and diluted at 1 : 100 in 0·05% Tween 20/TBS. Filters were washed 3 times in 0·05% Tween 20/TBS, and incubated with secondary antibody-alkaline phosphatase (AP) conjugated γ-chain specific goat antihuman IgG (Sigma) diluted at 1 : 30 000 in 0·05% Tween 20/TBS. Reactivity was developed with AP substrate (cat 170–6432, Bio-Rad) to locate positive plaques. After eight sequential rounds of experiments, 20 filters were screened by antibody probes.

Positive phage stock preparation

The filters were aligned with the plates to select each positive plaque. These were transferred to microcentrifuge tubes containing 200 µl of 1 × lambda dilution buffer. A drop of chloroform was added and the tube vortexed briefly. Phage was eluted at 4°C overnight and centrifuged at 10 000 r.p.m. (8000 × g) for 2 min to remove debris. Phage stock was prepared according to the manufacturer's instructions; briefly, titred positive plaques were plated at 104 pfu on LB agarose containing 10 m m MgSO4. Following incubation at 37°C for 5–7 h, 5 ml of 1 × lambda dilution buffer was added, and the plates incubated at 4°C overnight. A few drops of chloroform were added to the plate and swirled briefly. The liquid containing phage lysate from the plate was poured into a sterile 50-ml polypropylene tube. Two ml of chloroform was added to the tube, which was then vortexed for 2 min, and centrifuged at 10 000 × g for 10 min. Supernatants were stored at 4°C for up to three months.

Isolation of phage DNA from positive plaques

To the collection of positive plaque supernatants, DNase I (Sigma) to 1 µg/ml and RNase A (Sigma) to 5 µg/ml, were added and incubated at room temperature for 30 min. Chloroform was added to a final concentration of 5%, and the tubes vortexed for 30 s, followed by centrifugation at 10 000 × g for 10 min at 4°C to pellet the debris. The aqueous phage was transferred to new centrifuge tubes and mixed with an equal volume of 20% PEG (polyethylene glycol)/2·0 m NaCl, incubated on ice overnight and centrifuged at 10 000 × g for 15 min at 4°C. The supernatant was discarded and the isolated phage DNA identified as a grayish smear on the side of each tube.

DNA extraction

The phage pellet was resuspended in 500 µl of 1 × lambda dilution buffer. To the phage DNA, EDTA to 20 m m, SDS to 0·5%, and proteinase K (Sigma) to 50 µg/ml final concentration were added. Following incubation at 65°C for 1 h, an equal volume of phenol:chloroform was added, and mixed by gentle inversion for 10 min. The mixture was then centrifuged at 7200 × g for 10 min at room temperature. The supernatant was collected and the procedure repeated until the interface was clean (usually one extraction was sufficient). The above steps were repeated with chloroform only to remove any residual phenol (the small amount of aqueous material left at the interface was collected separately in 1·5-ml microcentrifuge tubes, centrifuged, and combined with the larger fraction). 1/10 volume of 3 m NaOAc and 2·5 volumes of 95% ethanol were added and the preparation held at −20°C overnight to allow DNA precipitation. The DNA pellet was collected by centrifugation at 20 000 × g for 15 min and washed with 70% ethanol for 5 min at room temperature. The pellet was allowed to dry until the edges turned clear; it was then resuspended in 50 µl of TE (tris/EDTA) buffer and stored at 4°C or visualized on a 0·7% agarose gel in TAE (tris/acetate/EDTA) buffer with ethidium bromide after electrophoresis at 100 V for 40 min.

Insert size determination by PCR and DNA sequencing

Five µl of λ phage samples or 1 µl of DNA (≈ 20 ng) extracted from λ phage was utilized for PCR reactions with 100 n m each of forward primer 5′-AGGCACATGGCTGAA TATCGA-3′ and reverse primer 5′-CCAGACCAACTGG TAATGGTAGC-3′ in Hotstar Taq MasterMix (Qiagen, Australia). The PCR conditions were: 95°C for 10 min for one cycle for initial enzyme activation, followed by 40 cycles of 95°C for 15 s for denaturation and 60°C for 5 min for annealing and extension using a FTS-320 Thermal Cycler (Corbett Research, Sydney, Australia). PCR products (0·6–1·3 kb) were purified by Wizard PCR preps DNA Purification System (Promega Corporation, Annandale, Australia), and visualized on a 0·7% agarose gel in TAE buffer with ethidium bromide after electrophoresis at 100 V for 40 min. DNA sequencing reactions were performed using the forward primer on the Applied Biosystems ‘Big-Dye’ Terminator (vers. 3) chemistry (Westmead Hospital, Sydney, Australia). The results were analysed with BLAST (Standard nucleotide-nucleotide BLAST search tool program) via the National Centre for Biotechnology Information (NCBI) BLAST network service.

Recognition of identified antigens for individuals by dot blots

Preparation of E. coli lysates for absorption of anti-E. coli sera. E. coli strain Y1090r was grown to stationary phase (OD600 = 1·4) in 100 ml aliquots in LB medium. Cells were harvested by centrifugation at 4000 × g for 10 min at 4°C and washed 3 times with PBS. Cells were resuspended in 3 ml of 50 m m borate buffer (pH 8·0); the suspension was frozen and thawed 5 times, and sonicated at full power for 6 periods of 20 s each on ice using a Branson Sonifier 450 (Branson Ultrasonics, USA). The extract was clarified by centrifugation at 15 000 × g for 10 min at 4°C. The supernatant (containing 8 mg protein/ml) was transferred to a clean tube and stored at – 20°C up to two months. E. coli proteins were bound to NHS-activated Sepharose 4 Fast Flow beads (Pharmacia) according to the manufacturer's instructions. Briefly, 4 ml of a 50% (v/v) slurry was washed on a scintered glass filter with 100 ml of 1 m m HCl. The gel was suspended with an equal volume (2 ml) of 50 m m borate buffer (pH 8·0), and 1 ml E-coli extract (8 mg protein) added. The mixture was incubated by end-over-end rotation overnight at 4°C. Non-reacted groups on the matrix were blocked by tris buffer (pH 8–9) for 4 h at 4°C using end-over-end rotation. This was followed by washing with tris buffer (pH 8–9) and acetate buffer (pH 3–4) alternatively for 3 times. The bound matrix was resuspended in an equal volume of 0·05% Tween 20 in TBS and aliquoted into 36 vials. To deplete E. coli reactive antibodies, patient sera (n = 22), control subject sera (n = 12) and one reference control and one blank were added to final concentrations of 1 : 50 dilution. The sera were incubated with rotation overnight at 4°C. The absorbed antibodies were ready for use and were stored at 4°C in the presence of 0·05% sodium azide until used for immunodetection.

For immunodot blots, positive plaque stock antigens diluted in TBS to a concentration of 1 µg protein/spot, were applied to prewetted nitrocellulose membranes (0·45 µm, 9 × 12 cm size, Bio-Rad) and filtered in a vacuum microfiltration apparatus (96-well format, Bio-Rad). Blotted antigens were blocked with 1% BSA/TBS. All membranes were immunodetected by preabsorbed patient sera depleted of anti-E. coli (Y1090r) reactivity (n = 22), and clinically healthy subject sera similarly depleted (n = 12). The secondary antibody employed was alkaline phosphatase (AP) conjugated γ-chain specific goat antihuman IgG (Sigma) diluted 1 : 30 000 in 0·05% Tween 20/TBS. IgG antibody binding antigen was visualized with AP substrate (cat 170–6432, Bio-Rad). Triplicate blots were performed for each experiment.

ELISA for anti-CD24 peptide antibody

Human CD24 immunodominant mature peptide [5] as ELISA antigen was synthesized by Chiron/Mimotopes (Melbourne, Australia) with N-terminal biotin followed by –SGSGSETTTGTSSNSSQSTSNSGLAPNPTNATTKA-OH (MW: 3514·90). CD24 peptide was pre-titrated to obtain an optimal working solution at 1 µm in 0·1% Tween 20/PBS (TPBS), added at 100 µl per well to streptavidin-coated microtiter plates (Mimotopes) and incubated for 1 h at 37°C followed by 3 washes with 0·05% Tween 20/PBS containing 0·1% sodium azide. Serial-four fold dilutions (from 1 : 10) of patient and control sera in TPBS in duplicate sets were incubated with the peptide for 1 h at 37°C and the wells were washed 3 times with TPBS; then incubated with secondary antibody-goat antihuman IgG (γ-chain specific) conjugated with alkaline phosphatase (AP) (Sigma) diluted at 1 : 30 000 in TPBS for 1 h at 37°C and washed as described above, with a final wash in PBS only. Bound conjugates were detected by p-Nitrophenylphosphate (PNPP) in AP buffer. The reactions were stopped by addition of 1 m NaOH after overnight incubation and measured at an optical density (OD) of 405 nm with an ELISA microplate reader (Bio-Rad Laboratories, Inc. Australia). The negative controls were performed with PBS in place of CD24 peptide, direct addition of conjugate to streptavidin-coated plates with biotin-CD24 peptide and nonrelated antibody as primary antibody.

Immunostaining of CD24

Sections of paired periodontitis sites and minimally inflamed gingival sites taken from each patient were used for probing with anti-CD24 antibody. Briefly, sections were fixed in acetone at 4°C for 10 min, treated with 0·3% hydrogen peroxide in methanol for 5 min to suppress endogenous peroxidase activity and blocked in 20% horse serum in phosphate buffered saline (PBS) for 1 h at room temperature. Sections were then incubated with monoclonal mouse antihuman CD24 (1 : 100; DAKO, Denmark) diluted in 10% foetal calf serum (FCS) in PBS for 1 h at room temperature in a humid chamber. Slides were washed in fresh PBS three times for 10 min each, with gentle agitation. Sections were incubated with goat antimouse antibody (1 : 100; DAKO, Denmark) conjugated with horseradish peroxidase for 1 h at room temperature, followed by three washes with PBS, then stained with enhanced diaminobenzidine (DAB) in stable peroxide buffer (Pierce Ltd). Sections were lightly counterstained with Mayer's haematoxylin and mounted. As negative controls, matched isotype monoclonal mouse IgG1 antibody (DAKO, Denmark) was used in place of the primary antibody. The immunohistochemical results obtained were consistent and reproducible.

Evaluation of results

Gingival epithelial sections were coded and examined as four regions (Fig. 4a,b) for minimally inflamed gingiva (Min) and periodontitis (Perio) sites, respectively, that is, oral epithelium (OE), gingival crest (GC), gingival sulcus (GS) and gingival attachment (GA) or pocket epithelium (PE). To ensure reproducibility, serial sections from both sites were processed in batches under uniform, standardized conditions. The intensity of immunostaining was assessed on a visual analogue scale ranging from 0 to 3, that is, 0 = − (negative), no staining or very little was detectable; 1 = +, weak staining in the cell membrane were observed; 2 = + +, moderate staining; 3 = + + +, strong staining. In some sections, the staining was variable over the length of the OE or PE, therefore an average score 1·5 or 2·5 was recorded. All sections were coded and scored by two independent observers who were calibrated to reference slides.

Fig. 4
Photomicrographs of staining patterns for CD24. (a, b) The low magnification ( × 20) photomicrographs show the four epithelial regions of minimally inflamed gingiva (Min) and periodontitis (Perio). Intense (+++) staining is observed in gingival ...

Wilcoxon matched pairs test (Prism Software, San Diego, CA, USA) was performed to compare all regions in both sites from the same patients. Also analyses of ELISA plots and linear regression were calculated by Mann–Whitney test and Spearman correlation (Prism Software, San Diego, CA, USA), respectively. A level of P < 0·05 was accepted as statistically significant.


Identification of cross-reactive epithelial antigens

Positive plaques from the phage library were identified by immuno-screening using pooled patient sera previously absorbed with host E. coli lysate (Fig. 1). Eleven positive plaques were identified from 20 filters. The inserts were sequenced and amongst these, 6 positive clones were found to encode protein fragments corresponding to polypeptides expressed by the library (Fig. 2). After searching GenBank four antigens (CD24, LDH-A, AOP2 and NFAT5) and products of novel genes encoded in chromosome 1 and chromosome 18 were identified. Semi-quantitative immunodot blot analysis of individual sera indicated that CD24 was strongly recognized by 4 patient and 2 control subject sera. LDH-A and NFAT5 antigens were each strongly recognized by sera from 4 patients. AOP2 antigen was recognized by 5 patients. Chromosome 1 and chromosome 18 novel gene products were strongly recognized by 8 patient, and 2 control subject sera and 5 patient and 3 control subject sera, respectively. The results are summarized in Table 1.

Fig. 1
One of the filters displaying positive plaques screened by pooled patient sera previously absorbed with E. coli strain Y1090r lysates.
Fig. 2
Insert size determination by PCR and DNA sequencing analysis. Eleven selections were sequenced, and 6 positive clones (0·6–1·2 kb) were found to encode for protein fragments corresponding to polypeptides expressed by the library. ...
Table 1
Identification of auto-antigens by cDNA library screening with periodontitis patient and control subject sera IgG.

In relation to the potential significance of cell surface expression of CD24, this antigen was selected for further study.

Titration of anti-CD24 peptide antibody by ELISA

Peptide ELISA assays were established by using biotinylated CD24 peptide antigen attached to streptavidin-coated microtiter plates. Optical density values were corrected by subtracting the mean background interference for raw data including the no-antigen control run in parallel wells. Intra-assay variation for each sample was measured as the average OD deviation. The antibody titres were calculated by plotting absorbance OD at 405 nm (y-axis) versus four fold antiserum dilutions (x-axis) using the average for each duplicate set, then estimated as the reciprocal of the dilution corresponding to the mid-point binding of saturation binding curves. Where overnight incubation produced a net OD max of <0·5, a titre of <10 was assigned.

Association of anti-CD24 peptide antibody with periodontal disease indices

Patients with periodontitis were divided into two subgroups; mild-moderate disease activity and severe disease activity, according to diagnosis score (1–4, 5–6), selected periodontitis sites for pocket depth (4–6, 7–11 mm) and attachment loss (5–8, 9–11 mm) (Fig. 3). Titres of anti-CD24 IgG in the clinically healthy gingiva (CHG) subjects were not significantly different from the disease subgroups. The mean titres of anti-CD24 IgG in two subgroups segregated on the basis of diagnosis score were not significantly different. However, for the disease indices of pocket depth and attachment loss, there were significantly greater titres in patients with mild-moderate disease than in those with more severe disease activity (P < 0·05) by Mann–Whitney test, a finding confirmed by Spearman correlation (pocket depth: r = −0·50, P < 0·05; attachment loss: r = −0·54, P < 0·05), and a negative trend for diagnosis score (r = −0·32, P > 0·05).

Fig. 3
Scatter plots showing serum anti-CD24 IgG titres of clinically healthy gingiva (CHG) and periodontitis subgroups relative to periodontal disease indices. Horizontal bars represent the mean titres of anti-CD24 IgG. Titres ≤10 (under the broken ...

Association of anti-CD24 peptide antibody with periodontal disease risk factors and classifications

Data indicated a positive trend (n = 22, r = 0·31, P > 0·5) between increase in age and anti-CD24 IgG titres for the periodontal disease group. There was no correlation found in clinically healthy subjects. There were no significant differences for anti-CD24 IgG between smokers (n = 9) and nonsmokers (n = 13) grouped for disease indices. However, there was a negative correlation for anti-CD24 IgG in generalized periodontitis patients related to diagnosis score (n = 14, r = −0·54, P < 0·05). Titres of anti CD24 antibody were significantly greater (P < 0·01) in patients with generalized periodontitis (n = 14) compared with localized periodontitis (n = 8) where titres were consistently <200 (data not shown).

Immunostaining patterns for CD24

CD24 reactivity was detected in the four regions of both periodontitis sites and minimally inflamed gingival sites (Fig. 4a,b). The major staining was noted at the cell surface along the cell membrane in the spinous layers and suprabasal layers (Fig. 4c,e). Staining was also observed in the pocket epithelium (Fig. 4b,d,f) and gingival attachment (Fig. 4a) with an intense reaction. Although the patterns of staining for CD24 were similar in both biopsies from the same patient, the levels of CD24 immunoreactivity in the four regions of the diseased biopsies were higher than levels in the minimally inflamed biopsies[OE (P < 0·01), GC (P < 0·05) and GA/PE (P < 0·01)] (Fig. 5).

Fig. 5
Scatter plots showing the grade of CD24 staining in the four regions of epithelium in both of minimally inflamed gingiva (Min) and periodontitis (Perio) tissues. Horizontal bars represent the mean grade of each group. There were differences in the OE ...


In this study, 4 auto-antigens (CD24, AOP2, LDH-A and NFAT5) and peptide products of uncharacterized genes encoded on chromosome 1 and chromosome 18, were identified by screening a phage library. Although these antigens have not yet been studied in the context of periodontitis, CD24, AOP2 and LDH-A are known to play important roles in epithelial cell responses related to cancer and infectious diseases.

Lactate dehydrogenase A, a tetrametic gene product, is expressed in human retinoblastoma cell lines [18], human corneal endothelial cells [19], breast cancer cell lines [20] and endothelial cells [21]. Lactate dehydrogenase A activity is a key point in the stimulatory effect of epidermal growth factor (EGF) on lactate production in cultured Sertoli cells [22]. This gene has not previously been reported to be expressed in keratinocytes. It also occurs in some bacteria, including streptococci, Actinomyces and Lactobacillus spp. [23].

Peroxiredoxin 6 (PRDX6) (also called antioxidant protein 2, or AOP2) is a novel peroxiredoxin family member whose function in vivo is unknown. There is evidence that AOP2 is a novel thiol-dependent antioxidant that functions to scavenge particular hydroperoxides in the cell and mediate specific signals [24]. PRDX6 is widely expressed in most tissues, being particularly abundant in epithelial cells [25,26]. The PRDX6 knock-out mouse model has enabled demonstration of the importance of this molecule in protection against damaging oxidizing species [25].

The functions of NFAT5 under isosmotic conditions present in vivo remain unknown. NFAT5 is necessary for optimal cell growth ex vivo under conditions associated with osmotic stress [27]. Cells are protected from the osmotic effect of high levels of sodium ion and urea by accumulating compatible osmolytes such as sorbitol, betaine, and myo-inositol. These osmolytes are involved in maintaining cell volume and electrolyte contents because they do not perturb protein structure and function over a wide range of concentrations. Sorbitol is produced by the reduction of glucose by aldose reductase, while betaine and myo-inositol are transported into the cells through specific transporters. Under hyperosmotic stress, transcription of genes encoding these proteins is strongly induced [28]. NFATs are targets of α6β4 integrin signalling and are involved in promoting carcinoma invasion, highlighting a novel function for this family of transcription factors in human cancer [29].

In paired biopsies, almost all of the epithelial cells within the epithelial attachment of minimally inflamed specimens were reactive with anti-CD24 antibody, while uniform strong reactivity was detected for the pocket lining epithelium of periodontitis biopsies. This was a consistent finding in the 22 patients studied. Preferential expression within the different gingival domains could relate to the developmental origin of the epithelial cells [30]. In this context both the epithelial attachment and pocket epithelium have cytokeratin profiles that are similar to developing epithelial tissues [30] that demonstrate extensive expression of CD24. There is evidence that both phorbol esters and interferon gamma can induce CD24 expression in epithelial cells [10] indicating that proximity to the inflammatory focus could also influence CD24 expression by epithelia.

Intracellular calcium concentrations are significantly increased when lymphocytes are treated with an anti-CD24 antibody [31], a finding of interest in relation to the capacity of calcium ions to modulate significantly the proliferation and differentiation of keratinocytes [32].

CD24 expression in these critical epithelial domains could be significant in the context of functioning as a costimulant for T cell activation [33]. It is of interest that CD24 has recently been reported to have a critical function in the expansion and persistence of auto-reactive T cells in foci of autoimmune pathology [34]. This has important potential implication for the generation of auto-reactive antibodies in the local tissue site of the periodontium. In this context, Rajapakse and Dolby [35] reported evidence for production of auto-reactive antibodies to collagen within gingival tissues. Of further interest is the reported relationship of CD24 polymorphism to risk for, and progression of, multiple sclerosis [36]. Heterogeneity of CD24 is also imparted by tissue-specific glycosylation patterns [37].

It is noteworthy that serum IgG antibody titres to CD24 peptide correlated with more favourable clinical status within the periodontal disease group. While there is a danger of over-interpreting a causal effect from this association, the observation is of interest. For some established autoimmune diseases, titres of auto-reactive antibodies correlate with disease. Anti-endothelial antibodies were related to disease activity in systemic lupus erythematosus [38]. Similarly, anti-desmoglein 1 protein correlated with clinical severity of pemphigus foliaceus [39]. Conversely, pneumococcal vaccines may protect against atherosclerosis by cross-reactivity of anti-pneumococcal polysaccharide antibodies with oxidized lipoprotein, masking macrophage recognition of these deposits within the artery wall [40].

Further investigation of the relationship of cross-reactivity to streptococcal antigens [4] and the effect of antibodies recognizing CD24 on keratinocyte function, will be informative.


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