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Oral Epithelial Overexpression of IL-1[Alpha] Causes Periodontal Disease

Posted on: Wednesday, 19 January 2005, 03:00 CST

ABSTRACT

Periodontal disease is a bacterial infection that results in inflammatory destruction of tissues that support the teeth, including connective tissue and bone. In this study, we report that transgenic mice that overexpress the 17-kDa form of IL-1α in the basal layer of oral mucosal epithelium develop a syndrome that possesses all of the cardinal features of periodontal disease, including epithelial proliferation and apical migration, loss of attachment, and destruction of cementum and alveolar bone. In this model, bacterial colonization and infection were not required, since levels of periodontal bacteria were equivalent in transgenic and wild-type mice, and continuous treatment with antibiotics from birth did not ameliorate the disease. Our findings therefore indicate that elevated levels of IL-1α in the oral microenvironment can mediate all of the clinical features of periodontal disease.

KEY WORDS: interleukin-1α, periodontitis, inflammation, transgenic, mucosa.

Received July 29, 2003; Last revision July 29, 2004; Accepted August 2, 2004

INTRODUCTION

Periodontal disease is characterized by inflammatory destruction of tissues that support the tooth, including the epithelial attachment, periodontal ligament, cementum, and alveolar bone, often culminating in tooth loss. Although bacterial infection is required for periodontitis, less than 20% of the variability in periodontal disease expression can be accounted for by the presence or absence of bacterial plaque, indicating a significant contribution of the host response to disease expression (Kornman and Le, 2000; McDevitt et al., 2000).

Of the host factors evaluated, the expression of interleukin-1 is most closely linked to periodontal disease progression. Gingival crevicular fluid levels of IL-1 are elevated in periodontitis, and decrease following treatment (Masada et al., 1990). Tissue levels of IL-1β are elevated at sites of progressive periodontitis (Stashenko et al., 1991). Concomitant antagonism of IL-1 and TNF inhibited periodontal destruction in non-human primates (Assuma et al., 1998; Graves et al., 1998), an effect more recently shown to be attributable solely to the inhibition of IL-1 (Delima et al., 2002).

We have previously reported the generation of two lines of transgenic mice, TgIL-1.1 and TgIL-1.2, that overexpress IL-1α in basal keratinocytes under the control of the keratin 14 (K14) promoter (Groves et al., 1995). The high-expressing TgIL-1.1 mice were found to exhibit spontaneous inflammatory skin lesions, a wasting syndrome, and impaired fertility. The lower-expressing TgIL- 1.2 mice exhibited a similar but milder phenotype.

Because keratin 14 is also expressed by oral keratinocytes, we determined the effect of overexpression of IL-1α by keratinocytes on the periodontium. We report that IL-1α overexpression results in a syndrome with all of the cardinal features of periodontitis, suggesting that oral keratinocyte- derived IL-1α may be a primary contributor to the disease process in humans.

MATERIALS & METHODS

Generation of Transgenic Animals

The generation and characterization of the IL-1α transgenic animals, designated TgIL-1.1 and TgIL-1.2, were reported previously (Groves et al., 1995). Tg-IL 1.1 had a higher expression of IL- 1α and more exaggerated skin disease compared with a milder phenotype in TgIL-1.2. For these studies, TgIL-1.2 mice and background strain control mice (FVB/N and C57B1/6) were used. All mice were bred and maintained at the Harvard Institutes of Medicine Animal Facility. Animals were age-, sex-, and strain-matched with littermate controls.

Immunohistochemical Analysis for Transgene Expression

Animals were killed at various ages by CO2 asphyxiation. Tongue, palatal, buccal mucosal, and skin specimens were isolated, fixed in methacarn (60% methyl alcohol, 30% chloroform, 10% acetic acid), and embedded in paraffin. Five-micrometer sections were prepared and stained for IL-1α with rabbit polyclonal antisera.

Figure 1. IL-1α levels are increased in the oral mucosa of TgTL-1.2 mice. (A) Immunohistochemical staining of tongue sections of wild-type (left) and TgIL-1.2 (right) mice with anti-IL-1α antibodies. (B) Protein levels of IL-1α in gingiva are elevated 47-fold in TgIL-1.2 mice (n = 8) compared with wild-type (n = 8). Data are presented as mean SEM (p < 0.0001).

Figure 2. (A) Alveolar bone loss is increased in TgIL-1.2 mice. Representative samples of de-fleshed mandibles. Arrows denote exposed cementum and indicate the extent of attachment loss. (B) Kinetic analysis of periodontal bone loss in IL-1α transgenic and wildtype mice. By 10 wks of age, IL-1α transgenic mice (n = 4) exhibited a 1.6-fold increased bone loss compared with wild-type mice (n = 4) (p < 0.001 ). This difference was increased to 3.6- fold after 31 wks (n = 5) (p < 0.0001). Antibiotic treatment failed to reduce bone loss in IL-1α transgenics at 10 wks (n = 4) and 1 8 wks (n = 4) of age. Data are presented as mean area of bone loss SEM.

Gingival Cytokine Levels

Tissue surrounding the maxillary teeth was isolated, minced, weighed, and digested in a mixture of 500 U/mL collagenase, 100 g/ mL bovine serum albumin, 100 g/mL Zwittergent-12, 50 g/mL gentamycin, and 10 mM Hepes buffer in RPMI1640. Following sonication and centrifugation, the supernatant was stored at -70C until tested for IL-1α by a commercially available ELISA assay (Biosource, Camarillo, CA, USA). Results were expressed as pg cytokine/mg gingival tissue.

Microbiological Analysis

Hemisected mandibles of 42-week-old mice were isolated, and the molar region was crushed in a sterile mortar and pestle, diluted with pre-reduced anaerobic sterilized transport media, sonicated, serially diluted, and cultured on blood agar plates under anaerobic conditions. Colony-forming units were counted after 5 days.

Measurement of Alveolar Bone Loss

Mandibles were de-fleshed in a dermisted beetle colony, bleached, and mounted on microscope slides. The lingual surfaces of the mandibular first and second molars were examined at 25 magnification with the use of a stereomicroscope and a computer imaging system (Optimus, Bioscan, Richmond, WA, USA). We determined the extent of horizontal bone loss by measuring the area enclosed by the cemento- enamel junction (CEJ), the alveolar bone crest, and the mesial and distal aspects of the tooth as described previously (Niederman et al, 2001).

Antibiotic Treatment

Pregnant IL-1α transgenic and control (FVB/N) mothers were placed on a defined diet without or with antibiotics (metronidazole, amoxicillin, bismuth; BioServ, Frenchtown, NJ, USA) three days prior to parturition, as described previously (Niederman et al., 2001). Pups were maintained on the same diet until death. This regimen has been shown to prevent periodontal disease progression in P- and E- selectin-deficient mice (Niederman et al., 2001).

Histomorphometry of Periodontal Tissues

For histomorphometry, mandibles (n = 4/group) were decalcified in EDTA, embedded in paraffin, and sectioned at 7- intervals. Every fifth section was mounted and stained with H & E. The two most centrally located sections, relative to the long axis of the molar teeth and the interdental papilla, were analyzed histomorphometrically with the use of Adobe Photoshop and NIH Scion Image analysis software. Pocket depth was measured as the vertical distance from the tip of the interdental papilla to the site of epithelial attachment to the tooth. Attachment level and alveolar bone height were measured with reference to a horizontal line drawn between the furcae of the first and second mandibular molars. The cross-sectional area of the interdental papillae occupied by epithelium was also determined.

Statistical Analysis

Data were reported as mean standard error or standard deviation. Student's t test was used for comparisons between groups.

Table. Histopathologic Features of Periodontal Disease in IL- 1α Transgenic Mice

Figure 3. Periodontal histopathology in 42-week-old wild-type (A) and IL-1α transgenic (B) mice. High-power view of interdental area between the mandibular first and second molars from wild-type mice (A) shows normal periodontal tissues. IL-1α transgenic mice (B) show severe periodontal disease, with extensive epithelial proliferation, pocket formation, apical migration of epithelium, attachment loss, and reduced alveolar crestal bone height extending below the furcae of the molars. T, tooth roots; B, alveolar bone; E, gingival epithelium. Arrows indicate site of epithelial attachment. Horizontal bars: 50 .

RESULTS

IL-1α Expression in Oral Tissues

FVB/N mice heterozygous for the IL-1α transgene under the control of the keratin 14 promoter (TgIL-1.2 line) and non- transgenic littermates were analyzed for the effects of transgene expression on oral tissues. Tissue samples obtained from the tongue, palate, and buccal mucosa of transgenic mice, but not non- transgenic littermates, demonstrated strong immunoreactivity for IL- 1α (Fig. 1A). Gingival tissues from TgIL-1.2 mice contained levels of IL-1α that were 47 times higher than in non- transgenic littermates, as determined by ELISA (Fig. 1B).

Histopathologic Features of Perio\dontitis in IL-1α Transgenic Mice

Alveolar bone resorption is a cardinal feature of periodontal disease (Lindhe et al., 1980). At all time points analyzed (10 to 31 wks), IL-1α transgenic mice demonstrated increased alveolar bone loss compared with age- and sex-matched non-transgenic control mice (Figs. 2A, 2B). Differences between transgenic and non- transgenic littermates increased with age. This finding was background-strain-independent, since equivalent bone loss was noted when the K14/IL-1 transgene was expressed in C57B1/6 (data not shown).

Importantly, other histopathologic characteristics of the periodontal lesions in IL-1α transgenic mice were consistent with the features typical of human periodontal disease (Lindhe et al., 1980). As shown in Fig. 3B, older IL-1α transgenic animals had extensive proliferation of the gingival epithelium, which occupied the entire interdental papilla, at the expense of the underlying connective tissue. This proliferation resulted in a nearly six-fold increase in the area occupied by epithelium in the transgenic mice (Table). Other changes included significant increases in periodontal pocket depth and decreases in the level of epithelial attachment to the tooth, both of which are consistent with the apical migration of the epithelium during the disease process. There was no cementum seen coronal to the site of epithelial attachment, suggesting that cemental resorption had occurred concurrent with the epithelial migration. Finally, the height of the alveolar crestal bone was significantly reduced, confirming the direct observations made above (Fig. 2). Taken together, these findings are consistent with the well-established histopathology of periodontitis in humans and in other animal models. Unlike bacterial-induced periodontal lesions, however, the lesions of the IL-1α transgenic mice were marked by both the relative absence of bacterial plaque and only a modest gingival inflammatory infiltrate.

Role of Indigenous Microbiota in Periodontal Pathology

Bacterial infection is considered to be the most important environmental factor in human periodontal disease (Socransky and Haffajee, 1997; Haffajee and Socransky, 2000). We therefore asked whether the susceptibility of TgIL-1.2 mice to bone loss was the result of increased oral microbial colonization. The results indicated clearly that levels of both aerobic and anaerobic microbial flora were comparable between transgenics (n = 7) and wild- type littermates (n = 9) (aerobic CFU [mean SEM]-wild-type, 69,500 5500; TgIL-1.2, 86,200 11,700; anaerobic CFU-wild-type, 102,000 21,500; TgIL-1.2, 119,500 23,300; no significant differences observed). This suggests that the periodontal disease observed in the transgenic animals was not the result of an increased bacterial load in the oral cavity.

To determine whether periodontal disease in IL-1α transgenics could be prevented by reducing the oral flora, we fed animals an antibiotic-containing diet (metronidazole, amoxicillin, bismuth) from birth until death after 10 and 18 wks of age (Niederman et al., 2001). Despite continuous antibiotic treatment, IL-1α transgenic mice demonstrated the same level of periodontal disease as untreated animals, indicating that the observed pathology was independent of bacterial infection (Fig. 2B).

DISCUSSION

IL-1 has been strongly implicated as a key final common pathway mediator in periodontal disease. Analysis of our data demonstrated that transgenic mice expressing high levels of IL-1 1α in oral mucosal epithelium develop a syndrome with all the cardinal features of severe periodontitis, including attachment loss, increased periodontal pocketing, and resorption of cementum and bone. This pathology could not be attributed to a greater intra-oral bacterial load, since bacterial counts were equivalent in transgenic and non- transgenic mice, and reduction of the oral flora by continuous treatment with antibiotics did not influence the development of disease. To our knowledge, this is the first demonstration that cytokines produced by epithelial cells in the oral micro- environment can cause a periodontitis-like syndrome.

Oral keratinocytes serve both as a mechanical barrier to invasion by pathogens and as a source of pro-inflammatory mediators, such as IL-1α. In skin, normal epidermal epithelium contains large amounts of IL-1α as well as pro-IL-1β (Hauser et al, 1986; Kupper et al., 1986). One potential role of this intracellular reservoir of IL-1 is the rapid initiation of inflammatory and repair processes following injury or infection (Dinarello, 1994; Kupper and Groves, 1995). In this regard, IL-1 primes neutrophils for increased phagocytosis and bactericidal activity (Yagisawa et al., 1995). Keratinocytes constitutively produce IL-1, but can be further induced to synthesize additional IL-1 as well as multiple secondary cytokines and chemokines (Groves et al., 1995). In turn, IL-1 induces widespread gene expression, including molecules that have been implicated in periodontal disease pathogenesis: adhesion molecules, chemokines, cytokines, cyclooxygenase 2 (COX-2), inducible nitric oxide synthetase (iNOS), metalloproteinases, and other proteolytic enzymes (Kupper, 1989; Larsen et al, 1989; Meikle et al, 1989; reviewed in O'Neill and Greene, 1998).

A critical feature of IL-1 is its ability to stimulate bone remodeling through activation of osteoclasts as well as osteoclast precursors (Jimi et al, 1996). IL-1 is the most potent mediator of bone resorption in vertebrates (Stashenko et al, 1987). In experimental models of periodontal disease, animals treated with IL- 1 blockers have demonstrated a 67% reduction in the number of osteoclasts, as well as a 60% reduction in bone loss (Delima et al, 2002). It is of interest that most of the leading risk factors in periodontal disease, including smoking, diabetes, genetics, osteoporosis, and stress, have been associated with elevated tissue and gingival crevicular fluid (GCF) levels of IL-1 (Johnson and Organ, 1997; Salvi et al, 1998; Deinzer et al, 1999; McDevitt et al., 2000).

IL-1 has also been implicated in the pathogenesis of osteoporosis (Pacifici, 1996). A functional IL-1 signaling pathway was found to be necessary for systemic bone loss induced by ovariectomy in a mouse model of osteoporosis (Lorenzo et al, 1998). Preliminary data from our laboratory corroborate these findings by indicating that TgIL-1.2 mice are also susceptible to osteoporosis of the vertebrae and long bones (Dayan and Stashenko, unpublished findings).

We considered the possibility that periodontal bone loss in K14/ IL-1α transgenic mice was simply a localized manifestation of systemic bone loss. However, we believe that this is unlikely. First, other cardinal histopathologic features that characterize authentic periodontal disease in humans-including an increase in periodontal pocketing, apical migration of the epithelial attachment along the root surface, loss of periodontal ligament, and disruption of its attachment to cementum-were also seen in TgIL-1.2 mice, in addition to the resorption of cementum and bone (Lindhe et al., 1980). In addition, studies of ovariectomy-induced osteoporosis in mice reported only minimal periodontal bone loss (Moriya et al., 1998), and furthermore these tissues did not exhibit the multiple histologic features of periodontal disease observed in our model.

In human periodontal disease, pathogenic micro-organisms initiate an inflammatory cascade that culminates in tissue destruction. In a subset of individuals, severe disease occurs in the absence of a large bacterial load, suggesting an exaggerated response to a modest microbial challenge (Reinhardt et al., 1993). By creating a model in which IL-1 is produced constitutively in the gingival micro- environment, we appear to have effectively bypassed the infectious component of periodontitis. This conclusion, however, must be qualified by the possibility that the residual oral flora remaining after antibiotic treatment could still be necessary, in conjunction with elevated levels of IL-1α, for the observed bone loss. Additional studies in gnotobiotic animals are necessary for this issue to be definitively resolved.

Mice deficient in both P- and E-selectin also develop severe early-onset periodontal disease, secondary to impaired entry of leukocytes into tissues, including the oral cavity (Niederman et ai, 2001). The defect in cell migration in P/E^sup -/-^ results in increased colonization of bacterial pathogens in the oral cavity, as well as increased IL-1α expression in gingival tissues. An antibiotic regimen (amoxicillin, metronidazole, bismuth) identical to the one used in the present study successfully prevented periodontal bone destruction in P/E-selectin-deficient mice, whereas it had no effect on either the magnitude or kinetics of periodontal disease in K14/IL-1α mice. As noted, P/E-selectin-deficient mice have ten-fold elevated levels of oral microbial colonization compared with wild-type littermates, levels which were reduced to wild-type levels by this antibiotic regimen (Niederman et al., 2001). Interestingly, overall numbers of oral bacteria were not reduced in wild-type animals, suggesting that while superinfection with pathogens is reduced by this regimen, the 'normal' oral flora is not substantially affected, at least numerically.

Taken together, these findings support the concept that IL-1 is a central downstream mediator of the infectious process, resulting in loss of epithelial attachment and bone resorption. Targeting IL-1 signaling pathways for inhibition may be an attractive therapeutic objective in the treatment of human periodontitis, and can be tested with the IL-1α transgenic overexpression model.

ACKNOWLEDGMENTS

The authors thank Patrice Tully and Dr. Hajime Sasaki for assistance with histomorphometry and D\r. Ulrike Spate for help with graphics. This work was supported by grants AR-40124, AR-42689, DE- 09018, and DE-11664 from the NIH.

REFERENCES

Assuma R, Oates T, Cochran D, Amar S, Graves DT (1998). IL-1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis. J Immunol 160:403-409.

Deinzer R, Forster P, Fuck L, Herforth A, Stiller-Winkler R, Idel H (1999). Increase of crevicular interleukin 1 beta under academic stress at experimental gingivitis sites and at sites of perfect oral hygiene. J Clin Periodontol 26:1-8.

Delima AJ, Karatzas S, Amar S, Graves DT (2002). Inflammation and tissue loss caused by periodontal pathogens is reduced by interleukin-1 antagonists. J Infect Dis 186:511-516.

Dinarello CA (1994). The biological properties of interleukin-1. Eur Cytokine Network 5:517-531.

Graves DT, Delima AJ, Assuma R, Amar S, Oates T, Cochran D (1998). Interleukin-1 and tumor necrosis factor antagonists inhibit the progression of inflammatory cell infiltration toward alveolar bone in experimental periodontitis. J Periodontol 69:1419-1425.

Groves RW, Mizutani H, Kieffer JD, Kupper TS (1995). Inflammatory skin disease in transgenic mice that express high levels of interleukin 1 alpha in basal epidermis. Proc Natl Acad Sci USA 92:11874-11878.

Haffajee AD, Socransky SS (2000). Microbial etiological agents of destructive periodontal diseases. Periodoniology 5:78-111.

Hauser C, Saurat JH, Schmitt A, Jaunin F, Dayer JM (1986). Interleukin 1 is present in normal human epidermis. J Immunol 136:3317-3323.

Jimi E, Ikebe T, Takahashi N, Hirala M, Suda T, Koga T (1996). Interleukin-1 alpha activates an NF-kappaB-like factor in osteoclast- like cells. J Biol Chem 271:4605-4608.

Johnson GK, Organ CC (1997). Prostaglandin E2 and interleukin-1 concentrations in nicotine-exposed oral keratinocyte cultures. J Periodontal Res 32:447-454.

Kornman KS, Le H (2000). The role of local factors in the etiology of periodontal diseases. Periodontology 2:83-97.

Kupper TS (1989). Mechanisms of cutaneous inflammation. Interactions between epidermal cytokines, adhesion molecules, and leukocytes [published erratum appears in Arch Dermatol 125:1643, 1989]. Arch Dermatol 125:1406-1412.

Kupper TS, Groves RW (1995). The interleukin-1 axis and cutaneous inflammation. J Invest Dermatol 105:62S-66S.

Kupper TS, Ballard DW, Chua AO, McGuire JS, Flood PM, Horowitz MC, el al. (1986). Human keratinocytes contain mRNA indistinguishable from monocyte interleukin 1 alpha and beta mRNA. Keratinocyte epidermal cell-derived thymocyte-activating factor is identical to interleukin 1. J Exp Med 164:2095-2100.

Larsen CG, Anderson AO, Oppenheim JJ, Matsushima K (1989). Production of interleukin-8 by human dermal fibroblasts and keratinocytes in response to interleukin-1 or tumour necrosis factor. Immunology 68:31-36.

Lindhe J, Liljenberg B, Listgarten M (1980). Some microbiological and histopathological features of periodontal disease in man. J Periodontol 51:264-269.

Lorenzo JA, Naprta A, Rao Y, Alander C, Glaccum M, Widmer M, et al. (1998). Mice lacking the type I interleukin-1 receptor do not lose bone mass after ovariectomy [see comments]. Endocrinology 139:3022-3025.

Masada MP, Persson R, Kenney JS, Lee SW, Page RC, Allison AC (1990). Measurement of interleukin-1 alpha and -1 beta in gingival crevicular fluid: implications for the pathogenesis of periodontal disease. J Periodontal Res 25:156-163.

McDevitt MJ, Wang HY, Knobelman C, Newman MG, di Giovine FS, Timms J, et al. (2000). Interleukin-1 genetic association with periodontitis in clinical practice. J Periodontol 71:156-163.

Meikle MC, Atkinson SJ, Ward RV, Murphy G, Reynolds JJ (1989). Gingival fibroblasts degrade type I collagen films when stimulated with tumor necrosis factor and interleukin 1: evidence that breakdown is mediated by metalloproteinases. J Periodontal Res 24:207-213.

Moriya Y, Ito K, Murai S (1998). Effects of experimental osteoporosis on alveolar bone loss in rats. J Oral Sci 40:171-175.

Niederman R, Westernoff T, Lee C, Mark LL, Kawashima N, UllmanCullere M, et al. (2001). Infection-mediated early-onset periodontal disease in P/E-selectin-deficient mice. J Clin Periodontol 28:569-575.

O'Neill LA, Greene C (1998). Signal transduction pathways activated by the IL-1 receptor family: ancient signaling machinery in mammals, insects, and plants. J Leukocyte Biol 63:650-657.

Pacifici R (1996). Estrogen, cytokines, and pathogenesis of postmenopausal osteoporosis. J Bone Miner Res 11:1043-1051.

Reinhardt RA, Masada MP, Kaldahl WB, DuBois LM, Kornman KS, Choi JI, et al. (1993). Gingival fluid IL-1 and IL-6 levels in refractory periodontitis. J Clin Periodontol 20:225-231.

Salvi GE, Beck JD, Offenbacher S (1998). PGE2, IL-1 beta, and TNF- alpha responses in diabetics as modifiers of periodontal disease expression. Ann Periodontol 3:40-50.

Socransky SS, Haffajee AD (1997). The nature of periodontal diseases. Ann Periodontol 2:3-10.

Stashenko P, Dewhirst FE, Peros WJ, Kent RL, Ago JM (1987). Synergistic interactions between interleukin 1, tumor necrosis factor, and lymphotoxin in bone resorption. J Immunol 138:1464- 1468.

Stashenko P, Fujiyoshi P, Obernesser MS, Prostak L, Haffajee AD, Socransky SS (1991). Levels of interleukin 1β in tissue from sites of active periodontal disease. J Clin Periodontol 18:548-554.

Yagisawa M, Yuo A, Kitagawa S, Yazaki Y, Togawa A, Takaku F (1995). Stimulation and priming of human neutrophils by IL-1 alpha and IL-1 beta: complete inhibition by IL-1 receptor antagonist and no interaction with other cytokines. Exp Hematol 23:603-608.

S. Dayan1, P. Stashenko2, R. Niederman2, and T.S. Kupper1*

1 Department of Dermatology, Brigham and Women's Hospital, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Room 671, Boston, MA 02115, USA; and 2 Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA; * corresponding author, tskupper@rics.bwh.harvard.edu

J Dent Res 83(10):786-790, 2004

Copyright American Association for Dental Research/American Academy of Implant Dentistry Oct 2004

Source: Journal of Dental Research

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