Abstract
Periodontal disease is a significant cause of tooth loss among adults. It is initiated by pathogenic bacteria, which trigger an inflammatory response that is effective in preventing significant microbial colonization of the gingival tissues. In some individuals, the reaction to bacteria may lead to an excessive host response, resulting in periodontal tissue destruction. Recent developments suggest that interleukin (IL)–1 genetic polymorphisms may identify certain individuals who have a predisposed susceptibility to periodontal breakdown and that elevated levels of IL-1 are found in individuals with periodontal disease. However, there is no direct evidence that IL-1 per se is responsible for the critical events that occur in periodontitis. We investigated the role of IL-1 in periodontal disease in a Macaca fascicularis primate model, using human soluble IL-1 receptor type I as a specific inhibitor. The results indicate that inhibition of IL-1 alone significantly reduces inflammation, connective tissue attachment loss, and bone resorption that are induced by periodontal pathogens
Periodontitis is an infectious disease that results in the destruction of the structures that support the teeth, namely, alveolar bone and connective tissue attachment to teeth. It is estimated that 15%–35% of the adult population of the United States suffers from this multifactorial disorder, which is a significant cause of tooth loss in this population [1, 2]. Periodontal disease is initiated by bacterial colonization of the tooth surface that triggers an immunoinflammatory response in the adjacent host tissues. Although bacterial pathogens are required to initiate the disease process, it has become evident that their presence alone is not sufficient to cause the degree of tissue destruction that occurs in periodontitis [2, 3]
Interleukin (IL)–1 is a proinflammatory cytokine that has a large array of biological functions and directly regulates several genes expressed during inflammation. There are 2 principle forms of IL-1 that have agonist activity (IL-1α and IL-1β) and a third ligand (IL-1 receptor antagonist) that functions as a competitive inhibitor [4]. IL-1α and IL-1β have similar biological activities, which are conferred by their ability to bind to high-affinity receptors on the surface of target cells, designated “IL-1 receptor type I” (IL-1RI) and “IL-1 receptor type II” (IL-1RII) [4–7]. IL-1RI and its coreceptor, known as “IL-1 receptor accessory protein,” are responsible for signal transduction in IL-1–responsive cells [5, 6]. IL-1RII, which has a minor intracellular domain, has been reported to function principally as a decoy receptor [7], although there are reports that it can mediate cellular activity [8]. IL-1RII can be cleaved from the cell surface at sites of inflammation to function as an endogenous inhibitor of IL-1 [7]
The biological effects of IL-1 have been implicated in the pathogenesis of several pathologic conditions, including asthma [9], congestive heart failure [10], arthritis [11], septic shock [12], and preterm labor [13]. There are several lines of evidence that implicate IL-1 in the pathogenesis of periodontal disease. IL-1 is produced in several types of host cells found in the periodontal environment, in response to periodontal pathogens or their products, such as lipopolysaccharide [14, 15]. Moreover, IL-1 levels are high at sites at which periodontal disease is present [16, 17]. Recently, it has been reported that genetic polymorphisms at the IL-1 gene locus may identify individuals who are susceptible to periodontitis, although there is disagreement about which variant of the IL-1 gene confers susceptibility [18–20]
To determine whether the expression of IL-1 per se is a significant factor in bacteria-induced periodontitis, experiments were carried out in which IL-1 activity was inhibited by treatment with soluble IL-1RI (sIL-1RI) in a nonhuman primate model of experimental periodontitis. The results indicate that inhibition of IL-1 significantly reduces the progression of inflammation toward alveolar bone and the destructive effects of periodontal pathogens
Materials and Methods
Experimental designThe human sIL-1RI used in these experiments was generously provided by Immunex and is described in [21]. Animal experiments were approved by the Institutional Animal Care and Use Committee at the Boston University Medical Center. A Macaca fascicularis model of periodontal disease was used in which silk ligatures were tied around the posterior teeth to induce accumulation of plaque and the initiation of periodontitis. This model is considered to be superior to other animal models because it demonstrates clinical and histologic features similar to the disease state in humans and because periodontal destruction is clearly triggered by bacterial infection [22]. Nine animals were studied (age range, 3–6 years). Three animals were killed at the zero time point to provide baseline measurements. Experimental periodontitis was induced in the remaining 6 animals by tying silk ligatures inoculated with Porphyromonas gingivalis (strain A7436) around the posterior mandibular teeth, as described elsewhere [23]. Three animals (the experimental group) received injections of sIL-1RI (6 μg per injection) to the gingiva of the posterior teeth, and 3 animals (the control group) received injections of sterile PBS. Each injection was placed locally in the interdental papillae 3 times weekly for a total of 6 weeks
Specimen preparationBlock sections of the mandibular posterior sextants were dissected and fixed in 4% paraformaldehyde for 2 days at 4°C. The interproximal areas between the first and second premolars and the second premolar and first molar of the right and left sides of the mandible were decalcified by immersion in cold Immunocal (Decal) for 2 weeks and prepared for cryostat sectioning. Sections were cut parallel to the long axis of the teeth at the midline of the interdental area and stained with hematoxylin-eosin
Histomorphometric analysisMononuclear inflammatory cells were identified as round, deeply blue hematoxylin-stained cells with condensed nuclei and little cytoplasm and were easily distinguished from elongated fibroblastic cells, histiocytes, and endothelial cells. The inflammatory front was defined as the fields nearest alveolar bone that had a minimum of 10 inflammatory cells per field at high magnification (×500), as we described elsewhere [24]. Both the distance between the field closest to alveolar bone and the bone and the mean distance between the fields of the inflammatory front and alveolar bone were measured using a computer-assisted image-analysis system. The number of inflammatory cells in close proximity to bone was measured as described in [25]. The loss of connective tissue attachment was defined as the distance from the cementoenamel junction to the most coronal extent of the connective tissue attachment. Bone loss was calculated by subtracting the distance between a line connecting the 2 opposing cementoenamel junctions and the most coronal extent of the bone crest at the zero time point from the average distance between these points for the 6-week period. Histologic landmarks are shown in figure 1. Histomorphometric analysis was performed using Image ProPlus software (Media Cybernetics). Measurements were performed by a single blinded examiner, and values were calculated from those results. Approximately 25% of the specimens were randomly selected for reexamination, and the results were generally within 15% of the original value
Histology of the periodontium in health and disease. A Normal periodontium, with histologic features designated by arrows. B Effect of periodontal disease on histologic landmarks of the periodontium. Note the decrease in the length of the connective tissue attachment and the reduction in bone height that are caused by periodontal disease
Statistical analysisHistomorphometric data were calculated for each block (6 blocks per group). Statistical significance was determined by analysis of variance, using Scheffe’s multiple comparison as a post hoc test. P<.05 was considered to be significant
Results
Periodontal disease is initiated by bacteria, which colonize the tooth surface and trigger an inflammatory response that is thought to be responsible for the ensuing tissue damage. An important feature is the progression of an inflammatory infiltrate from an area subjacent to the epithelium toward the alveolar bone. To establish the relationship between the induction of periodontitis and the appearance of an inflammatory infiltrate, the distance between the inflammatory front and the alveolar bone surface was measured for each specimen (figure 2). In the group of animals with periodontitis that did not receive treatment, this distance was considerably less than that in the zero-time-point group. However, when animals were treated with sIL-1RI, the “migration” of the inflammatory infiltrate toward alveolar bone was significantly less. This reduction was 55% for the mandibular premolars and 70% for the premolar/first molar samples. When the closest aspect of the inflammatory front to alveolar bone was assessed, reductions of 60% and 80% were observed (figure 2C and 2D). All of the differences were significant
Soluble interleukin-1 receptor type I (sIL-1RI) inhibits the progression of an inflammatory front toward alveolar bone in experimental periodontitis. Periodontal disease was initiated in Macaca fascicularis primates. The control group (no treatment) received injections of vehicle alone (PBS), and the experimental group received injections of IL-1 antagonists. Periodontal disease was not initiated in the zero-time-point group, which provided baseline measurements. The mean distance between the fields of the inflammatory front and alveolar bone and the distance between the aspect of the inflammatory front closest to the alveolar bone surface and bone were measured, as described in Materials and Methods. A Distance between the inflammatory front and bone for mandibular premolars; B distance between bone and the aspect of the inflammatory front closest to bone for the premolar and the first molar; C distance between the inflammatory front and bone for the mandibular premolars; D distance between bone and the aspect of the inflammatory front closest to bone for the premolar and the first molar
The degree of inflammation within the deep connective tissue was also assessed by measurement of the presence of inflammatory cells adjacent to alveolar bone (figure 3). In the mandibular premolar samples, the number of inflammatory cells was significantly reduced (by 70%) in animals treated with sIL-1RI. For premolar/first molar samples, treatment with sIL-1RI reduced the number of inflammatory cells by 50%, which was also statistically significant
Soluble interleukin-1 receptor type I (sIL-1RI) inhibits inflammatory cell recruitment close to alveolar bone during periodontal destruction. Periodontal disease was initiated in Macaca fascicularis primates in a control group (no treatment) and an experimental group (treated with sIL-1RI). The no. of inflammatory cells in the connective tissue adjacent to alveolar bone was measured as described in Materials and Methods. Measurements were made between the mandibular premolars and between the premolar and first molar
Histologic analysis was undertaken to determine the effects of sIL-1RI on destruction of the supporting periodontium. Loss of connective tissue attachment represents a reduction in the length of dense connective tissue that is firmly attached to teeth. At the zero time point, only minimal loss of attachment had occurred, whereas considerable loss was evident when periodontal disease was initiated (figure 4). Treatment with sIL-1RI was effective in reducing this connective tissue loss; a 30% reduction was noted in the premolar sections, and a 70% reduction was seen in the premolar/first molar region. Both differences were statistically significant
Soluble interleukin-1 receptor type I (sIL-1RI) inhibits the loss of connective tissue attachment in experimentally induced periodontitis. Periodontal disease was initiated in Macaca fascicularis primates (B and C) and periodontal tissue was compared to that of primates in the control group (A). (Original magnification, ×10.) Animals that had periodontitis received treatment with vehicle (PBS) alone (B) or sIL-1RI (C) for 6 weeks. The upper arrows in panels A–C indicate the original coronal extent of the connective tissue attachment, denoted by the presence of cementum, and the lower arrows indicate the coronal extent of the connective tissue attachment at the time that the animal was killed. The length of connective tissue attachment was assessed in histologic sections by image analysis. Measurements were made between the mandibular first and second premolars (D) and between the second premolar and first molar (E)
Treatment with sIL-1RI also reduced the loss in height of alveolar bone (figure 5). In both the control and experimental groups, there was a loss of bone height; however, the loss of alveolar bone height was less in animals that received injections of sIL-1RI than it was in control animals. This decrease was statistically significant and ranged from 65% for the mandibular premolars to 60% for the premolar/molar area
Application of soluble interleukin-1 receptor type I (sIL-1RI) inhibits loss of alveolar bone height. Periodontal disease was initiated in Macaca fascicularis primates. The experimental group received injections of sIL-1RI, and the control group (no treatment) received injections of vehicle alone (PBS). The loss of alveolar bone height was determined histologically as described in Materials and Methods. Measurements were made between the mandibular premolars and between the premolar and first molar
Discussion
Periodontal disease is caused by selected species of bacteria that colonize the tooth surface and invade the adjacent tissue, causing inflammation and, ultimately, connective tissue destruction and bone resorption. That antibiotics effectively inhibit experimental periodontitis in several animal models, including the primate model used here, provides evidence for the central role of bacteria in triggering the destructive effects [2, 22]. However, evidence that tissue loss caused by periodontal disease is reduced by inhibitors of prostaglandins and inhibitors of matrix metalloproteinases indicates that much of the tissue damage is caused by up-regulation of the host response [26, 27]
IL-1 plays a critical role in stimulating the innate host response and in this capacity prepares the host to defend itself against bacteria [28]. The importance of IL-1 in protecting the host from oral pathogens was demonstrated in a murine model in which the dental pulp was infected with bacteria in mice lacking IL-1RI [29]. Surprisingly, osteolysis was greater in mice lacking functional IL-1RI than it was in wild-type mice, a result that is opposite to the findings of the present study. This is likely the result of the difference in the model. In the endodontic model, the host response must be activated from a naive state, whereas in the periodontal model, the host response is already up-regulated by the presence of gingivitis, even before the initiation of periodontal disease [25]. Thus, in a naive host, IL-1 activity is needed to optimally induce a protective response to bacterial infection and thereby minimize soft- and hard-tissue loss, whereas further up-regulation under conditions in which an inflammatory response is already in place leads to enhanced destruction of connective tissue and osteolysis. This is consistent with previous reports that the beneficial effect of IL-1 is negated if its expression is excessive; for example, overproduction of IL-1 causes tissue destruction in humans with autoimmune diseases such as rheumatoid arthritis and also contributes to hypotension, hematologic dyscrasias, and septic shock [12]. In human clinical trials, IL-1 antagonists are therapeutically useful in treating arthritis [11]. Interestingly, blocking IL-1 appears to be more effective than blocking tumor necrosis factor (TNF) in reducing bone loss associated with arthritis. In animal models, it has been shown that inhibition of IL-1 is effective for treatment of a number of inflammatory conditions, including septic shock, experimental glomerulonephritis, and cerebral ischemia [12, 30, 31]
We have reported that simultaneous inhibition of IL-1 and TNF significantly reduces tissue destruction and inflammation in experimental periodontitis [24, 25, 32]. Because these studies used a combination of IL-1 and TNF inhibitors, the precise role of IL-1 was not defined. In addition, the recent focus on IL-1 as a marker of periodontal disease susceptibility and the effectiveness of IL-1 antagonist in halting bone loss caused by rheumatoid arthritis led us to focus specifically on the role of IL-1 in periodontal disease progression. The results indicate that treatment with sIL-1RI alone significantly reduces 3 critical parameters of periodontal tissue destruction: inflammation, loss of dense connective tissue attachment to teeth, and resorption of bone
The results presented here strengthen the notion that periodontal tissue loss is caused by an exaggerated host response to oral bacteria, as was first indicated by reports that inhibitors of prostaglandin synthesis reduce bone loss associated with experimentally induced periodontitis [26]. Our results also support the concept that a critical event in periodontitis is the progression of an inflammatory front from an area subjacent to the epithelium to deeper structures of the periodontium. The reasons behind this remain obscure. One mechanism may be acquisition by bacteria of the ability to penetrate deeper into the connective tissue. If this occurs, bacteria or their products, such as lipopolysaccharide, may induce expression of IL-1, which then plays an important role in the up-regulation of a host response that results in tissue destruction. The initiation of an inflammatory process through the induction of IL-1 would stimulate the production of secondary mediators, such as chemokines or cyclooxygenase products, which, in turn, would amplify the degree of inflammation [33, 34]. Once this occurs, induced matrix metalloproteinases would participate in the destruction of the connective tissue [27], while cytokines might reduce the capacity of the host to repair the damaged tissue through apoptosis of residents cells such as fibroblasts [35]. Finally, the induction of an inflammatory cascade stimulates osteoclastogenesis that results in destruction of bone [36]. The present data directly establish the crucial role of IL-1 in experimental periodontitis and highlight the potential use of IL-1R in the treatment of periodontal disease
Acknowledgment
The authors thank Alicia Ruff for administrative support in preparing the manuscript
References
Financial support: National Institute of Dental and Craniofacial Research (grant DEO 7559)
