A comparison of the impact of IL-1 in oral and dermal wound healing

Wound healing has three principal phases (^{Hunte et al., 1999}). The healing starts with an inflammatory phase stimulated by the production of cytokines and other inflammatory mediators. This is followed by a proliferation phase, particularly of fibroblasts and endothelial cells. This is then followed by a third phase, which involves the production and reorganization of an extracellular matrix leading to repair or regeneration. The inflammatory phase sets the stage for the repair process because it leads to the recruitment of leukocytes that produce growth factors and remove the debris of the wound. A number of inflammatory mediators are upregulated during the healing process, including interleukin-1 (IL-1). The importance of leukocyte recruitment has been shown in a variety of animal models. For example, mice with targeted deletion of P-and E-selectins exhibit reduced recruitment of inflammatory cells (neutrophils and macrophages) and impaired wound closure. Animals depleted of monocytes have diminished healing (^{Leibovich and Ross, 1975}). Thus, expression of inflammatory cytokines such as IL-1 may play a central role in the early events of wound healing, in part because they stimulate the recruitment of leukocytes.

Interleukin-1

Interleukin-1 stimulates many of the early events that are involved in the generation of an inflammatory response (^{Alheim and Bartfai,1998}). There are two IL-1 ligands with agonist activity, IL-1α and IL-1ß. Both bind to IL-1 receptors termed « type 1 » and « type 2 ». The type 1 IL-1 receptor (IL-1R1) is responsible for specific signaling. In most experimental situations the type 2 receptor has been shown to function as a non-signaling decoy receptor. Much that is known about the IL-1 receptor signaling has come from studies with mice that have targeted functional deletions of the IL-1 receptor type 1 (IL-1R1^_/_ that is IL-1 receptor ablated) (^{Glaccum et al., 1997}). These mice do not exhibit gross abnormalities, are resistant to LPS induced toxic shock and are capable of developing antibodies to exogenous antigen stimulation. In most but not all studies, mice with deficient IL-1 activity exhibit an attenuated inflammatory response as measured in the turpentine abscess formation model and are more susceptible to infectious agents (^{Leon et al., 1996} ; ^{Labow et al., 1997} ; ^{Yamada et al., 2000}).

Many different cell types are capable of producing IL-1α or IL-1ß. In healing tissue IL-1 is principally produced by epithelial cells (^{Matsumoto et al., 1997}) and has been postulated to accelerate epidermal healing (^{Saunder et al., 1990}). Although it is clear that proinflammatory cytokines such as IL-1 are expressed during wound healing, it is not known they ultimately promote or inhibit normal repair. Findings that IL-1 is overexpressed in wounds that heal poorly have cast doubt on the positive role of IL-1 in the healing process (^{Angele et al., 1999} ; ^{Trengove et al., 2000}). Further support for the concept that IL-1 expression is detrimental comes from studies indicating that IL-1 receptor antagonist can partially reverse the negative impact of tumor necrosis factor (TNF) on healing (^{Maish et al., 1999}). That wound healing is enhanced in mice that have a lower level of cytokine expression compared to normals also suggests that the expression of cytokines may inhibit repair even under normal conditions (^{Bettinger et al., 1994}).

IL-1 and oral healing

IL-1 expression is frequently thought of as a negative event. For example, its expression has been associated with a number of chronic diseases including arthritis and periodontal disease. IL-1 is elevated at sites with periodontal disease and is produced by several types in the periodontium (^{Hanazawa et al., 1985} ; ^{Jandinski et al., 1991} ; ^{Kinane et al., 1992}). Recent findings suggest that susceptibility to periodontal disease is influenced by genetic polymorphisms of the IL-1 gene (^{Kornman et al., 1997} ; ^{Laine et al., 2001} ; ^{Thomson et al., 2001}). However, there is disagreement on the specific variant of the IL-1 gene that is associated with enhanced susceptibility. Direct evidence for the role of IL-1 in periodontal disease comes from two experimental approaches. In one, IL-1 was applied to the gingiva in a rat model of periodontal disease. The application of exogenous IL-1 enhanced inflammation and bone resorption (^{Koide et al., 1995}). In the other, application of a monoclonal antibody to IL-1 was used to inhibit its activity in a primate model of periodontal disease. The inhibition of IL-1 reduced the migration of an inflammatory front toward alveolar bone and resulted in a diminished loss of connective tissue attachment and alveolar bone (^{Delima et al., 1994}).

We have examined the role of IL-1 in oral healing by comparing the closure of an excisional wound in mice deficient in the IL-1R1 compared to normal wild-type controls. A 1,5 mm punch biopsy was placed in the hard palate healing. Healing was significantly delayed in the IL-1 receptor deficient mice at several time points as measured by closure of the wound by healing connective tissue as well as by the healing epithelium (^{fig. 1}). For example, new connective tissue had covered approximately 40 % and new epithelium approximately 50 % of the original wound surface by day 4 in the wild type. A similar degree of healing was not seen until day 14 in the IL-1R^_/_ animals.

We previously reported that IL-1R^_/_ mice exhibited a limited capacity to resist infection by oral pathogens (^{Graves et al., 2000}). We therefore tested whether the delayed oral healing observed in the IL-1R^_/_ animals was due to a limited capacity to protect the wound site from infection. In these experiments, mice were treated with an antibiotic regimen that has been reported to substantially reduce the commensal flora in the oral cavity of mice (^{Baker et al., 1994}). The effect of antibiotic treatment was assessed by histomorphometric analysis of H E stained sections from mice sacrificed on day 14 (^{fig. 2}). Experimental mice treated with antibiotics had a significantly improved healing response. Epithelial and connective tissue wound closure was improved by 50 and 65 %, respectively, in the antibiotic treated group of IL-R^_/_ animals. It should be noted that healing in the wild type animals healing was completed at this time point with or without antibiotics.

Experiments were then undertaken to examine healing in experimental and wild type animals under conditions where bacterial challenge was less significant. To accomplish this, a 1,5 mm excisional wound was created in the scalp and the rate of healing determined (^{fig. 3}). At this site, the rates of healing in the wild type and control mice were similar except for epithelial healing at a single time point, day 7. This results points to a difference in the role that IL-1 plays in oral versus dermal healing.

Oral wound healing

It is typically thought that oral wound healing is fas-ter than healing in skin (^{Sciubba et al., 1978} ; ^{Yang et al., 1996} ; ^{Hakkinen et al., 2000}). Several factors have been proposed to explain this phenomena. For example, gingival fibroblasts have been reported to have properties that may facilitate healing (^{Tajima and Pimel, 1981} ; ^{Schor et al., 1996} ; ^{Lorimier et al., 1996}, ^{Lorimier et al., 1998} ; ^{Lee et Eun, 1999}). It has been reported that bacterial products, which are found in the oral cavity, can enhance wound healing (^{Kilcullen et al., 1998}). Saliva may also contribute to oral wound repair because it provides a moist environment and contains mediators that enhance repair (^{Mandel, 1987} ; ^{Yang et al., 1996}).

Despite the above assertions, there have been surprisingly few reports that have directly compared dermal versus oral healing under conditions where substantial regeneration of connective tissue is required. To investigate this issue we created 1,5 mm excisional punch biopsies in the scalp and hard palate of mice. Both of these wounds healed by formation of granulation tissue, extensive production of new connective tissue matrix and coverage by migrating epithelium. In addition, the wound was small enough so that contracture did not play a significant role in closing the wound. In most animals, the scalp healing progressed considerably faster than oral healing. For example, after seven days the epithelium had covered 96 % of the dermal wound surface and only 64 % of the same size biopsy of the palate. Similarly, the connective tissue had covered 95 % of the dermal wound surface and in the palate, only 48 % of the oral wound surface. Thus, by two histologic measures, oral healing was significantly delayed compared to a similar scalp wound, contrary to previous reports indicating.

The results of the above experiments indicate that the rate of healing is considerably delayed in oral versus dermal wounds. One reason that our results may differ from published studies is that we made an effort to compare healing of wounds that required a significant degree of new connective tissue formation, i.e. they were relatively deep. This may tend to leave the wound open to the environment for a longer period of time and as a result, render it more susceptible to inhibitory factors present in the oral environment. In previous studies, mucosal healing was emphasized that required relatively little new connective tissue formation compared to the type of excisional wound described here (^{Sciubba et al., 1978} ; ^{Yang et al., 1996} ; ^{Hakkinen et al., 2000}). Under the latter conditions, more rapid healing may occur because of positive influences in the oral environment. It is possible that the oral environment delays healing in wounds that remain open for a relatively long period of time. In contrast, the oral environment may enhance the rate of healing in wounds that close relatively quickly.

Conclusion

We have presented evidence that wound healing in an oral environment is different from that in a dermal environment. Unlike many previous studies we have tried to create wounds that have many similarities including a basal floor consisting of bone and the requirement for formation of a dense connective tissue matrix prior to epithelial bridging. The results indicate that the up-regulation of a proinflammatory cytokine such as IL-1 is considerably more important and oral environment that innate dermal environment. The present study addresses this issue and demonstrates that in a challenging environment, the loss of IL-1 receptor signaling causes a significant delay in excisional wound healing. In contrast, healing in the scalp is minimally affected by ablation of IL-1 receptors. This is due to the negative effect of bacteria on oral wound healing under conditions where the activity of IL-1 is deleted since treatment with antibiotics significantly restored the rate of healing in IL-1 receptor deficient mice. This interpretation is consistent with our previous finding that in a model of chronic infection, IL-1R1^_/_ mice have a diminished host response to endodontic pathogens (^{Chen et al., 1999}) and that cytokine activity is needed for the full antibacterial activities of PMNs. Moreover, histologic evidence suggested that wound exposure in IL-1R^_/_ mice could lead to a mild subclinical infection causing persistent inflammation and that in turn, impairs healing.

Acknowledgments

This work was supported by a grants from the NIDCR, DE07559(DG), DE11254(DG), DE07268(DG) and DE12482(SA).

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