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J Dent Res 83(9):683-687, 2004
© 2004 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Effects of Cyclosporin-A-induced Immunosuppression on Periapical Lesions in Rats

T. Kawahara1, S. Murakami2, Y. Noiri1,*, A. Ehara1, N. Takemura1, S. Furukawa2, and S. Ebisu1

1 Departments of Restorative Dentistry and Endodontology, and
2 Oral and Maxillofacial Radiology, Osaka University Graduate School of Dentistry, 1-8, Yamadaoka, Suita, Osaka 565-0871, Japan;

* corresponding author, noiri{at}dent.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclosporin A (CsA) might induce immune response alterations in periapical lesions and modify bone remodeling. This study determined the changes that occur in the periapical lesions of rats during CsA administration and after CsA withdrawal. After the induction of periapical lesions, the animals were treated with CsA (0–20 mg/kg/day) for 4 wks. Lesion volumes were measured by computed tomography. Histological observations and immunohistochemical evaluations were performed with anti-CD3 and anti-CD25 antibodies. CsA administration reduced lesion volumes, and the lesions significantly expanded after CsA withdrawal. CsA inhibited the proliferation and activation of T-cells at lesion sites. The effects of CsA on T-cells were dose-dependent up to 10 mg/kg/day, after which no significant difference was evident. These results suggest that CsA inhibits periapical destruction by interfering with T-cell function in periapical lesions.

KEY WORDS: periapical lesion • cyclosporin A (CsA) • rat • computed tomography • immunohistochemistry


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Survival rates of patients after organ transplantation have improved due to advances in immunosuppression. During periods of immunosuppression, infections in the oral cavity can worsen and spread to adjacent organs (Newman, 1996; Murray and Saunders, 2000). Patients scheduled to receive transplantations frequently undergo pre-therapeutic dental treatment to eliminate sources of dental infections (Meyer et al., 1999). The outcome of root canal treatments cannot be assessed immediately after treatment; therefore, success cannot be confirmed during the pre-therapeutic dental treatment period, and thus lesions might remain.

T-cells comprise a large portion of the inflammatory cells in periapical lesions and play an important role in lesion pathogenesis (Stashenko et al., 1994). Cyclosporin A (CsA), which can inhibit T-cell activation, is a clinically important immunosuppressive agent that is widely used for anti-rejection therapy (Calne et al., 1978). CsA might alter immune responses in periapical lesions. However, there is little information regarding any correlation between periapical lesions and CsA administration.

Computed tomography (CT) allows for non-invasive imaging and enables changes in lesion volume to be quantified over time; thus, it allows for a reduction in animal experiments compared with histological methods. The present study involved a CT scanning approach for the evaluation of the effects of CsA on rat periapical lesions.


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Periapical Lesions
Twenty five-week-old male Wistar rats, each weighing from 100 to 120 g, were used as animal models for periapical lesions. The use of the animals conformed to a protocol that was reviewed and approved by the Committee on Animal Research of the Osaka University Graduate School of Dentistry. The pulps of both mandibular first molars of all 20 animals were exposed to the oral environment for 4 wks, while periapical lesions were induced (n = 40) (Stashenko and Yu, 1989).

CsA Administration
The 20 animals were divided equally into 4 groups. CsA (Sandimmun®, NOVARTIS, Tokyo, Japan) was intraperitoneally injected into 3 groups of animals at daily doses of 5, 10, and 20 mg/kg/day, respectively, for 4 wks. The fourth group, controls, was injected with 0.9% NaCl. Following final injections, 3 rats per group were killed by pentobarbital overdose, and their periapical lesions (n = 6 per group) were evaluated histologically. CsA or 0.9% NaCl injections in the remaining 2 rats per group were halted; after 4 wks, the lesions (n = 4) were measured three-dimensionally and examined histologically after the animals’ death.

Lesion Volume Measurements
After 0, 1, 2, 4, and 8 wks (n = 10, 10, 10, 10, and 4, respectively) from the start of CsA administration, periapical lesions were measured. Rats were placed on a holder in the prone position, and the median planes of the animals were fitted to the z-axis of a CT scanning system (Light Speed Qx/i ver. 1.3, GE, Milwaukee, WI, USA). Micro-tomographic slices were acquired from each animal at 200-µm increments, covering the entire medial-lateral width of the mandible, and a series of coronal CT images was recorded. The areas of the periapical lesions were analyzed and measured by means of a CT image analysis system (Advantage WindowTM ver. 3.1, GE) (Fig. 1aGo). The volume of each lesion was defined as described (Kitai et al., 2002).



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  		Figure 1. Lesion volume measurements. (a) CT images analyzed by the image analysis system. (b) Data are expressed as the percentages of lesion volume at week 0 (weeks 0–4, n = 10; weeks 4–8, n = 4) and shown as means ± SD. Statistical analyses were performed by means of the Dunnett test. Significance (*) was determined at a level of p < 0.05.

 
Histopathological and Immunohistochemical Evaluations
The mandibles were dissected at 4 or 8 wks, fixed in periodate-lysine-paraformaldehyde fixative for 10 hrs, and decalcified in 10% EDTA containing 15% glycerol at 4°C. Eight-µm-thick serial frozen sections were prepared. Some of the sections were stained with hematoxylin-eosin, while others were stained immunohistochemically (Kawashima et al., 1996). Sections were incubated with anti-CD3 (1:500) (G4.18, Pharmingen, San Diego, CA, USA), anti-CD25 (1:2000) (OX39, Pharmingen), and anti-ED1 (1:5000) (ED1, Serotec LTD., Bicester, UK) antibodies, or stained with tartrate-resistant acid phosphatase (TRAP). Positive cell-counts were performed in the periapical area of 3 randomly selected fields (x 200) with the use of an ocular grid with 10 x 10 mm squares.

Statistical Analysis
The effects of CsA on periapical lesion volume were analyzed by the Dunnett test. Quantitative comparisons of the immunohistochemical assays were performed by Student’s t test. A p-value below 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Changes in Periapical Lesion Volume
During CsA administration, the lesion volumes of all the CsA-treated groups decreased up until week 4, while those of the control group increased slowly (Fig. 1bGo). Periapical lesion sizes decreased in a dose-dependent manner in the 5 and 10 mg/kg/day groups up until week 4. The difference between the 10 and 20 mg/kg/day groups was not statistically significant during the administration period. At week 4, lesion sizes of the 20 mg/kg/day group were statistically smaller than those of the 5 mg/kg/day and control groups, while the lesions of the 5 and 10 mg/kg/day groups were statistically smaller than those of the control group (p < 0.05).

At week 8, periapical lesion size expanded in all groups (Fig. 1bGo). The lesions in the 5 mg/kg/day group were not statistically different from those in the control group; however, the lesion volumes of the 10 and 20 mg/kg/day groups were statistically smaller than those of the control group (p < 0.05).

Histopathological Examination
At both weeks 4 and 8, abscesses with mixed inflammatory infiltrate were observed around the root apex (Figs. 2aGo, 2cGo, 2eGo, 2gGo), and the lesions were surrounded by fibroblasts (Figs. 2bGo, 2dGo, 2fGo, 2hGo). The extracellular matrix accumulation within the fibrous tissue was also slightly increased (Figs. 2bGo, 2dGo). At week 8, the peripheral morphology of the bone resorption in the lesions was more irregular in the 20 mg/kg/day group than in the control group (Figs. 2gGo, 2hGo).



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  		Figure 2. Pathological evaluation. (a,b) Control group at week 4. (c,d) 20 mg/kg/day group at week 4. (e,f) Control group at week 8. (g,h) 20 mg/kg/day group at week 8. Panels b, d, f, and h are high-magnification views of the periapical lesions shown in panels a, c, e, and g, respectively. AA, Apical Abscess; CE, Cementum; B, Bone. Scale bars: a, c, e, g = 250 µm; b, d, f, h = 50 µm.

 
Immunohistochemical Examination
At both weeks 4 and 8, CD3+ and CD25+ cells infiltrated the inflammatory tissues around the apical abscess (Figs. 3aGo–3dGo, 4aGo–4dGo). At week 4, infiltration of both the CD3+ and CD25+ cells had decreased in all the CsA-treated groups compared with the control group; however, a few CD25+ cells were seen in the 10 and 20 mg/kg/day groups (Figs. 3aGo–3dGo). The numbers of CD3+ and CD25+ cells significantly decreased in a dose-dependent manner in the 5 and 10 mg/kg/day groups compared with the control group, but there were no significant differences between the 10 and 20 mg/kg/day groups (Figs. 3eGo, 3fGo).



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  		Figure 3. Immunohistochemical staining at week 4. (a) Anti-CD3 staining of the control group. (b) Anti-CD3 staining of the 20 mg/kg/day group. (c) Anti-CD25 staining of the control group. (d) Anti-CD25 staining of the 20 mg/kg/day group. Scale bars = 100 µm. (e) CD3+ cell-counts in the lesions of each group (n = 6). (f) CD25+ cell-counts in the lesions of each group (n = 6). (g) ED1+ cell-counts in the lesions of each group (n = 6). (h) TRAP staining of the control group. (i) TRAP staining of the 20 mg/kg/day group. Values are the means ± SD. *A value of p < 0.01 was determined as statistically different in comparison with the controls.

 


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  		Figure 4. Immunohistochemical staining at week 8. (a) Anti-CD3 staining of the control group. (b) Anti-CD3 staining of the 20 mg/kg/day group. (c) Anti-CD25 staining of the control group. (d) Anti-CD25 staining of the 20 mg/kg/day group. Scale bars = 100 µm. (e) CD3+ cell-counts in the lesions of each group (n = 4). (f) CD25+ cell-counts in the lesions of each group (n = 4). (g) ED1+ cell-counts in the lesions of each group (n = 4). (h) TRAP staining of the control group. (i) TRAP staining of the 20 mg/kg/day group. Values are the means ± SD. *p < 0.05. **A value of p < 0.01 was determined as statistically different in comparison with controls.

 
At week 8, CD3+ and CD25+ cell infiltration increased in all the CsA-treated groups compared with that observed after 4 wks of CsA administration, while no obvious change was observed at either experimental point in the control group (Figs. 4eGo, 4fGo). The numbers of CD3+ cells in all the CsA-treated groups were significantly higher than in the control group, but no significant differences were found among the CsA-treated groups (Fig. 4eGo).

The numbers of ED1+ cells (Figs. 3gGo, 4gGo) and TRAP staining images (Figs. 3hGo, 3iGo, 4hGo, 4iGo) did not apparently differ between the experimental groups and the control group at weeks 4 and 8. In all groups, TRAP+ cells were localized at specific parts of the marginal bone around the periapical lesions.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Investigators have administered immunosuppressive agents to animals to block the function of target inflammatory cells to determine the roles of inflammatory cells in lesion pathogenesis, and periapical lesions were subsequently induced (Yamasaki et al., 1994; Waterman et al., 1998). Quantitative analyses of lymphocyte subpopulations consistently demonstrate an excess of T-cells over B-cells, indicating that, in both humans and in experimental lesions induced in rats, periapical granulomas are predominantly T-cell-type lesions. However, B-lymphocytes and plasma cells represent the major populations of periradicular inflammatory infiltrates (Márton and Kiss, 2000).

The IL-2 receptor {alpha}-chain, which can be identified with an anti-CD25 antibody with a heterotrimeric recognition structure, was expressed in activated T-cells (Caruso et al., 1997). The immunosuppressive effect on CsA was evaluated by quantitative analysis of CD25+ cells. Buchinsky et al.(1996) demonstrated that T-cell-depleted nude rats were resistant to the high turnover of osteopenia induced by CsA, while Sprague (2000) commented, in a review, that interactions between the immune system and bone turnover appeared to be important, since the effects of CsA seemed to depend on the presence of T-cells. CsA-induced interference with T-cell activation in rat periapical lesions, monitored through changes in CD25 expression, and the quantity of T-cells proliferating in the lesions were assessed with use of the anti-CD3 antibody. Close relationships were found between the results of the immunohistochemical analysis and the lesion volume measurements. After CsA withdrawal, proliferation and activation of T-cells were promoted in periapical lesions, suggesting reversibility of the agent activity. These results support the hypothesis that, among various kinds of inflammatory cells, T-cells play a crucial role in the development of periapical lesions.

Potential T-cell-mediated mechanisms relevant to periapical destruction—which activate the production of the bone-resorptive mediators IL-1, IL-6, and TNF{alpha}—stimulate the humoral immune response (Ishimi et al., 1990; Stashenko et al., 1994; Takeichi et al., 1996). CsA alters the immune response by selectively interfering with T-cell function, and has been shown to inhibit the bone-resorbing effects of IL-1 (Dawson et al., 1996). The inhibition of IL-1 activities might contribute, in part, to the decreased volume of periapical lesions observed during the CsA administration.

Many researchers have investigated the effects of CsA on the skeletal system, and conflicting findings have been reported. CsA prevented bone loss in adjuvant arthritis rats (del Pozo and Zapf, 1994) and inhibited vertebral bone resorption in weanling rats (Orcel et al., 1989). In contrast, severe bone loss was observed in CsA-treated rats (Movsowitz et al., 1988; Katz et al., 1994). The pathohistological findings of this study showed that CsA does not apparently influence mandibular bone structure.

To determine the effects of CsA on local immune responses, we examined macrophages and osteoclasts in the rat periapical lesions histochemically, using an anti-ED1 antibody (reactive to nearly all macrophages and dendritic cells) and TRAP staining. At week 4, the numbers of ED1+ cells and TRAP-staining images did not apparently differ between the CsA-treated groups and the control group, suggesting that CsA does not directly inhibit the destructive activities of the macrophages and osteoclasts.

It can be suggested that the decreased number of CD25+ cells did not influence the macrophage or osteoclast activities, and that there is no correlation between a reduction in lesion volume and the numbers of macrophages and osteoclasts. It is probable that the periapical lesion volume reduction observed was due not to changes in the numbers of macrophages and osteoclasts, but rather to decreased cytokine production by immune cells.

Within the limitations of the experimental design used, it is suggested that CsA would reduce tissue destruction at untreated or residual periapical lesion sites. A decreased periapical lesion volume, however, can be misinterpreted as healing. Root canal infection remains and might serve as a source of systemic dissemination. Therefore, a long-term follow-up study during and after CsA administration is desirable.


   ACKNOWLEDGMENTS
 
We thank Dr. Nobuo Noguchi for technical assistance, and Dr. Satoru Toyosawa and Dr. Yuzo Ogawa for technical advice regarding immunohistochemical staining. This study was supported by Grants-in-Aid for Scientific Research (Nos. 12557161 and 14207080) from the Japan Society for the Promotion of Science (JSPS), and via the 21st century COE program entitled "Origination of Frontier BioDentistry" at Osaka University Graduate School of Dentistry from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Received April 24, 2003; Last revision May 21, 2004; Accepted June 23, 2004


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Buchinsky FJ, Ma Y, Mann GN, Rucinski B, Bryer HP, Romero DF, et al. (1996). T lymphocytes play a critical role in the development of cyclosporin A-induced osteopenia. Endocrinology 137:2278–2285.[Abstract]

Calne RY, White DJ, Thiru S, Evans DB, McMaster P, Dunn DC, et al. (1978). Cyclosporin A in patients receiving renal allografts from cadaver donors. Lancet 2:1323–1327.[ISI][Medline]

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Dawson J, Hurtenbach U, MacKenzie A (1996). Cyclosporin A inhibits the in vivo production of interleukin-1beta and tumour necrosis factor alpha, but not interleukin-6, by a T-cell-independent mechanism. Cytokine 8:882–888.[ISI][Medline]

del Pozo E, Zapf J (1994). Skeletal growth and bone density as sensitive parameters in experimental arthritis: effect of cyclosporin A. Bone 15:625–628.[Medline]

Ishimi Y, Miyaura C, Jin CH, Akatsu T, Abe E, Nakamura Y, et al. (1990). IL-6 is produced by osteoblasts and induces bone resorption. J Immunol 145:3297–3303.[Abstract]

Katz I, Li M, Joffe I, Stein B, Jacobs T, Liang XG, et al. (1994). Influence of age on cyclosporin A-induced alterations in bone mineral metabolism in the rat in vivo. J Bone Miner Res 9:59–67.[ISI][Medline]

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