Levels of Interleukin-8 During Tooth Movement - Angle Orthodontist

Original Article

Levels of Interleukin-8 During Tooth Movement ¨ zmeric¸b; Cumhur Tuncera; ˙Idil Teomanb; Burcu C Burcu Balos¸ Tuncera; Nurdan O ¸ akılcıb; c d e Ays¸egu¨l Yu¨cel ; Reha Alpar ; Ko¨ksal Balos¸ Abstract: A host-derived neutrophil-activating cytokine interleukin-8 (IL-8) is secreted mainly by monocytes and is considered to be important in regulating alveolar bone resorption during tooth movement. The aim of this study was to evaluate the levels of IL-8 during mechanical forces on periodontal tissues at different stages of orthodontic therapy. Ten canine teeth of patients having different Angle classifications were selected for the study. After the premolars were extracted, the maxillary/mandibular canines were tipped distally. Gingival crevicular fluid was sampled from mesial and distal gingival crevices of each canine separately at baseline and one hour, 24 hours, six days, 10 days, and 30 days after the application of the force. An enzyme-linked immunosorbent assay for quantitative detection of IL-8 was used. Although there was an increase in the concentration of IL-8 at tension (mesial) sites after one hour, 24 hours, six days, and 10 days, a decrease was observed at 30 days. Pressure (distal) sites did not demonstrate such an increase at any period except at 10 days. However, the concentration of IL-8 at both sites showed a similar decrease and approached each other at day 30. We concluded that local host response toward the orthodontic forces might lead an increase in IL-8 and neutrophil accumulation, and this may be one of the triggers for bone remodeling processes. (Angle Orthod 2005;75:631–636.) Key Words: Cytokine; Interleukin-8; Tooth movement; Gingival crevicular fluid; Tension; Pressure

INTRODUCTION

The initial tooth movements require an acute inflammatory reaction with the migration of leukocytes. Experiments prove that neurotransmitters and cytokines are associated with the activation of alveolar bone and periodontal cells.2,3 Bone is resorbed in pressure areas on the side toward which the force is directed and deposited at the tensile areas in the contralateral position. This way, the movement of teeth through bone is facilitated.3–5 With moderate to severe forces, compression of the connective tissue fibers on the pressure side occurs, oxygen supplies and nutrition are restricted, and ischemia of the ligament begins. After several days of trauma, osteoclastic activity is seen, and the relatively healthy ligament cells observed near the necrotic tissue are available for repair activities. Undermining resorption results in clearance of the damaged tissue.6 Several studies have tried to explain the biological basis of tooth movement induced by mechanical stress. Tzannetou et al2 examined whether interleukin (IL)-1b and b-glucuronidase were present in the gingival crevicular fluid (GCF) of patients treated with rapid palatal expansion and stated that orthodontic/orthopedic forces might evoke changes in the levels of the inflammatory mediators in periodontal tissues. Besides, it has been reported that chemokines are reg-

Cytokines play an important role in intercellular signaling and have been implicated in the pathology of periodontal diseases, bone destruction, and bone response to orthodontic treatment. One of the most important breakthroughs of bone biology has been the identification of the role of cytokines in bone remodeling. Bone remodeling is used by orthodontists when forces transmitted to the surrounding tissues of the periodontium initiate the remodeling process.1 Research Assistant, Department of Orthodontics, Gazi University, Ankara, Turkey. b Associate Professor, Department of Periodontology, Gazi University, Ankara, Turkey. c Assistant Professor, Department of Immunology, Faculty of Medicine, Gazi University, Ankara, Turkey. d Professor, Department of Biostatistics, Hacettepe University Faculty of Medicine, Ankara, Turkey. e Professor, Department of Periodontology, Gazi University Faculty of Dentistry, Ankara, Turkey. Corresponding author: Burcu Balos¸ Tuncer, DDS, PhD, Department of Orthodontics, Gazi University, 84.sok, Ankara 06510, Turkey (e-mail: [email protected]) a

Accepted: July 2004. Submitted: May 2004. Q 2005 by The EH Angle Education and Research Foundation, Inc. 631

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¨ ZMERIC ¨ CEL, ALPAR, BALOS¸ TUNCER, O ¸ , TUNCER, TEOMAN, C ¸ AKILCI, YU

ulated during orthodontic tooth movements.1 The level of chemokines was significantly increased at the early stages and decreased in 7–10 days, and it was concluded that the early inflammatory response was the main trigger for bone remodeling processes.1 Stashenko et al3 examined the cytokine levels of IL-1a, IL1ß, and tumor necrosis factor-a in bone resorption and indicated that IL-1ß was an important mediator in the mechanism of bone resorption. Tooth movement leads to significant recruitment of cells belonging to the mononuclear phagocytic system.7 IL-8 is a potent proinflammatory cytokine that has a key role in the recruitment and activation of neutrophils during inflammation. It is secreted mainly by monocytes and is important in regulating alveolar bone resorption during tooth movement by acting early in the inflammatory response.8 The purpose of this study was to evaluate the levels of IL-8 during mechanically induced tooth movement and to evaluate the effect of orthodontic forces on periodontal tissues at tension and pressure sites.

Supragingival plaque was removed in conjunction with a record of the PI. Crevicular fluid was collected by isolating the area with cotton rolls, drying the teeth and adjacent marginal gingiva with air, and using paper strips (Periopaper-ProFlow Inc, New York) inserted for 30 seconds into the buccal crevice to a level of one mm below the gingival margin. After removing the first strip and waiting for one minute, a second strip was placed at the same site for another 30 seconds. Strips contaminated by saliva or blood were excluded from the sampled group. The paper strips from mesial and distal sites of each tooth were sealed in polypropylene containers separately. To determine the amount of GCF, an electronic scale (Precisa 62 A, Precisa instruments A6, CH-Dietikan, Switzerland) was used for weighing the paper strips before and immediately after the collection. The difference between the two weights gave the volume of fluid collected, assuming a specific gravity of approximately 1. Each sample was stored at 2708C until being assayed.

MATERIALS AND METHODS Ten canine teeth of adolescent patients (aged 15– 17 years) having different Angle classifications were selected for the study. All participants were systemically healthy and had not taken anti-inflammatory agents, antibiotics, immunosuppressants, or systemic contraceptives in the past six months. The participants’ rights were protected, and informed consent and assent were obtained according to the Gazi University Ethical Committee Board. The status of the periodontal tissues was determined by clinical periodontal assessments, including plaque index (PI), bleeding on probing (BOP), probing depth (PD), and clinical attachment level (CAL). Radiographic examinations were performed, and the smoking habits of all participants were also recorded. They all received a periodontal prophylaxis, including scaling and polishing before the collection of GCF. After the first premolars were extracted, the canines were distalized using 90 g forces for the mandibular canines and 115 g forces for the maxillary canines. The forces were generated by Ricketts sectional system arches and verified with a calibrated orthodontic force gauge.9 GCF from each tooth was collected at baseline (pretreatment observation period) and one hour, 24 hours, six days, 10 days, and 30 days after the application of the orthodontic force. During the examination periods no orthodontic activation was performed.

Cytokine analysis

GCF sampling GCF was sampled separately from the mesial and distal gingival crevices of each canine where the orthodontic forces were applied. Angle Orthodontist, Vol 75, No 4, 2005

Each strip was eluted twice with 100 mL of Hank’s balanced salt solution containing 0.5% bovine serum albumin by centrifugation (3000 3 g, 48C, 15 minutes). The kit for measuring IL-8 was Cyt Elisa (Cyt Immune Sciences Inc, College Park, Md). This kit is a sandwich enzyme immunoassay that measures the free forms of the cytokine IL-8. With the Cyt Elisa assay system, rabbit polyclonal antibodies generated against human IL-8 are used to capture human IL-8 in a sample. IL-8 present in the sample is bound to an anti–IL-8 monoclonal coating antibody. A second polyclonal antibody was added, and after incubation a colored product was formed in proportion to the amount of IL-8 present in the sample. The assay is observed using a streptavidin–alkaline phosphatase conjugate and an ensuing chromogenic substrate reaction. The amount of IL-8 detected in each sample was compared with an IL-8 standard curve that demonstrates a direct relationship between optical density and cytokine concentration. Optical density was measured at 450 nm by using ELISA Kit Reader (Sunrise, Tecan Austria GmbH, Gro¨ dig-Austria). The total amount of IL-8 was determined in picograms, and calculation of the IL-8 concentration in each sample was determined by dividing the amount of IL-8 by the GCF volume of each sample (pg/mL).2 Both the total amount and concentration of cytokine levels have been evaluated in other studies.10–12 Statistical evaluation The descriptive measurements (mean, median, standard deviation (SD), minimum and maximum val-

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IL-8 LEVELS DURING TOOTH MOVEMENT TABLE 1. Gingival Crevicular Fluid Volume at Mesial and Distal Sites of Teeth (mg) Groups

Statistics

Baseline

1h

24 h

6d

10 d

30 d

Mesial

Mean SD Median Minimum Maximum Mean SD Median Minimum Maximum

0.0013 0.0010 0.0010 0.0007 0.0034 0.0008 0.0005 0.0005 0.0002 0.0016

0.0010 0.0006 0.0008 0.0005 0.0024 0.0011 0.0006 0.0011 0.0004 0.0021

0.0017 0.0020 0.0006 0.0003 0.0074 0.0011 0.0006 0.0010 0.0003 0.0020

0.0016 0.0012 0.0017 0.0002 0.0036 0.0011 0.0005 0.0012 0.0003 0.0017

0.0008 0.0004 0.0008 0.0001 0.0014 0.0009 0.0004 0.0010 0.0003 0.0014

0.0024 0.0012 0.0023 0.0009 0.0037 0.0012 0.0004 0.0010 0.0008 0.0019

Distal

TABLE 2. Total Amount of Interkeukin-8 at Mesial and Distal Sites of Teeth (pg) Group

Statistics

Baseline

1h

24 h

6d

10 d

30 d

Mesial


1292.30 345.80 1244.15 911.14 1776.36 1407.30 590.50 1340.03 864.04 2440.78

1847.02 1425.90 1638.77 390.52 4114.20 1671.60 1478.30 1860.65 0.00 3201.86

1563.50 1129.50 1646.20 308.70 3234.10 1736.60 1341.50 1755.25 110.38 3516.72

2272.80 1408.40 3070.48 435.14 3586.14 1466.01 977.90 1682.16 393.16 2621.80

2012.50 1632.40 1724.30 504.56 4697.84 1606.70 915.80 1652.40 291.36 2780.42

1672.20 818.40 1498.70 970.64 3065.52 934.04 570.60 1017.74 278.96 1724.30

Distal

TABLE 3. Concentration of Interleukin-8 at Mesial and Distal Sites of Teeth (pg/ml) Groups

Statistics

Baseline

1h

24 h

6d

10 d

30 d

Mesial


1208.50 464.76 1208.46 522.45 1708.91 2635.80 1683.50 2417.77 824.35 4881.56

1706.40 883.10 1654.24 557.88 3050.97 1414.40 1502.70 1215.30 0.00 3853.57

1814.70 1211.20 1842.94 270.87 3219.90 1470.40 864.80 1418.76 157.68 2834.96

2139.90 1951.40 1806.16 543.92 5485.40 1299.60 589.30 1401.80 329.28 1914.61

3178.60 2164.20 2463.28 978.08 5864.80 1729.40 774.10 1986.01 934.14 2754.00

785.30 306.50 828.51 416.30 1078.49 726.40 383.00 679.16 278.96 1272.14

Distal

ues) are given in Tables 1 through 3. To understand the significant differences between the groups at a time interval, by taking the differences between two adjacent points, Mann-Whitney U-test was used. The results were also analyzed within each group by Wilcoxon test. RESULTS The clinical indices of subjects were recorded at baseline, and the mean and SDs of PI, PD, CAL, and BOP were measured as 1.172 6 0.37 mm, 2.124 6 0.535 mm, 2.190 6 0.624 mm, and 34.9 6 16.41 mm, respectively. No signs of periodontal destruction were observed in any subject. Tables 1 through 3 illustrate the mean 6 SD, median, minimum and maximum values of GCF volume,

total amount, and concentration of IL-8. The GCF volume was slightly greater at tension and pressure sites after 24 hours and 30 days than at the other observation periods (Figure 1). No statistically significant results were found between the groups (P . .05). The mechanical forces affected the amount of GCF differently in the tension and pressure sites. The total amount of IL-8 at both sites increased just after one hour of applying mechanical forces (Figure 2). Between the periods 24 hours and six days, increases occurred in the tension site (Figure 2). On day 6, it reached the maximum level at the tension sites, whereas the highest level at the pressure sites was observed in the first and 24th hour. No statistically significant results were determined between groups (P . .05). Angle Orthodontist, Vol 75, No 4, 2005

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FIGURE 3. Concentration of interleukin-8.

FIGURE 1. Distribution of total amount of gingival crevicular fluid by time.

volume, total amount, and concentration of IL-8 (P . .05). DISCUSSION

FIGURE 2. Distribution of total amount of interleukin-8 by time.

The concentration of IL-8 at the tension sites increased gradually until 30 days (Figure 3). At the pressure sites, no increase was demonstrated except at day 10. The values of IL-8 concentration at both sites showed a similar decrease and approached each other at day 30. IL-8 concentration among groups between baseline and the first hour was statistically significant (P , .05). Higher levels of IL-8 concentration were found at distal sites at baseline relative to the lesser amount of GCF volume at this site. No other statistically significant differences were determined between the periods within groups for GCF Angle Orthodontist, Vol 75, No 4, 2005

This study was designed to examine the levels of IL-8 during the initial phase of orthodontic tooth movement and to compare the amounts and concentrations of IL-8 between tension and pressure sites. The results clearly demonstrate that IL-8 production in the local tension site environment was greater than at the pressure sites, although the initial force on the first day led to a significant increase in IL-8 amount at both sites. Gianelly13 stated that excessive forces compressed the roots and caused undermining resorption. It was postulated that intermittent forces produced an increase in deposition of alveolar bone, whereas continuous loading was unable to stimulate the remodeling process.14 The interaction between mechanical forces and collagen turnover is important in tooth movement. There are several studies dealing with this subject.15– 17 These studies demonstrated that mechanical forces cause an increase in DNA synthesis, alkaline phosphatase activity, and collagen synthesis. The analysis of GCF is considered to be a useful method for examining cellular dynamics. 18–20 The method is noninvasive, and there are some advantages especially for human in vivo studies. At the tension site, an increase in the volume of GCF was determined in 24 hours in this study. On the other hand, at the pressure site it increased in the first hour and declined on day 10. At the fourth week, increase in GCF volume was observed at both sites. These results imply that mechanical trauma was responsible for the early inflammatory response of the

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IL-8 LEVELS DURING TOOTH MOVEMENT

tissues that was determined by the high levels of GCF volume. Experiments determining the activity of inflammatory mediators may reveal the rate of orthodontic tooth movement. Therefore, GCF can be useful in determining the optimum force amounts as well as the stabilization of biologic activities in the periodontium, which is considered important in retention of orthodontic treatment. IL-8 is believed to play an important role in the pathogenesis of various forms of periodontitis, and high levels were detected in such subjects.21,22 Because periodontal tissues are remodeled at both tension and pressure sites during tooth movement, the increased amount of IL-8 at both sites just after application of mechanical forces might be a sign of neutrophil reaction in the area. After the acute response, IL-8 stimulation might be continued at the tension sites. Therefore, under the influence of mechanical forces, pressure sites indirectly contribute to the production of IL8. Thus, we thought that the production of IL-8 is regulated differentially at tension and pressure sites and probably plays a major role in the initial stage of remodeling. Similar results were shown by Takahashi et al,23 who tried to clarify the hypothesis that the expression of matrixmetalloproteinase-8 and matrixmetalloproteinase-13 are regulated differently by tension and compression. Alternatively, because of the less active IL-8 production at pressure sites in this study, it was concluded that the initial force might be too heavy for a period and that decreased the cellular activity. A study was constructed to evaluate the effects of continuous and interrupted forces on mediators in vivo.24 Significant elevations of IL-1b levels were observed with a continuous and intermittent force at 24 hours. When a continuous force was applied, interleukin levels were decreased and maintained stable after 24 hours. Therefore, with an interrupted force, greater elevations were determined. They concluded that, intermittent forces have greater effects when cellular activity is considered. The intermittent force affected the amount of IL-8 in this study. Besides, IL-8 total amount showed highest levels in the pressure sites immediately in the first and 24th hour. This might be caused by an early upregulation of chemotactic activities directly after mechanical force application. This is in accordance with Davidovitch et al,1 who showed the acute inflammatory response in the initial phase of tooth movement. The increased production of the other mediators in tooth movement has been shown in several studies.25,26 At the tension sites, an increase was determined in the first hour and on day 6, and afterward the levels declined indicating the decrease in mediator activities.

Iwasaki et al27 reported that IL-1b levels fluctuated with a 28-day cycle. IL-8 may have the same cyclic periods, but we were not able to follow the IL-8 fluctuation for 30 days. A statistically significant difference in the concentration of IL-8 was found among groups in the baseline and first-hour periods (P , .05), which indicates the acute reaction. Thus, mediators are efficient in the early stages of inflammation and, therefore, the importance of the amount of initial force cannot be neglected. This finding is in accordance with Alhashimi et al,28 who stated that the early inflammatory reaction is responsible for the bone remodeling process. IL-8 concentration showed high levels at early stages and declined on day 30 at the tension sites. This gradual increase up to 10 days and apparent decrease afterward might indicate a cyclic period of activation of IL-8. At the pressure sites, a decrease was observed initially, and a slight elevation of the mediator was determined on day 10 and demonstrated low levels at day 30. CONCLUSIONS The health condition of the periodontium is important during orthodontic treatment and studies because forces in different directions, duration, and magnitudes cause changes in the periodontal tissues. Evaluation of these changes can be analyzed using GCF, which is a noninvasive and useful method. In this study, orthodontic forces evoked changes in the levels of IL-8 in periodontal tissues. The affect of mechanical stimulus on cellular activities demonstrated different levels of IL-8 at tension and pressure sites. This might be a trigger factor for bone remodeling processes. The initial orthodontic forces caused IL-8 levels to increase significantly after one hour and reached the highest level in the sixth day. The stabilization of the cellular activities should be determined in further studies. The mediator levels in GCF could possibly be used as reflecting parameters of periodontal status after orthodontic therapies in future. REFERENCES 1. Davidovitch Z, Nicolay O, Ngan P, Shanfeld J. Neurotransmitters, cytokines and the control of alveolar bone remodeling in orthodontics. Dent Clin North Am. 1988;32:411– 434. 2. Tzannetou S, Efstratiadis S, Nicolay O, Grbic J, Lamster I. Interleukin-1b and b-glucuronidase in gingival crevicular fluid from molars during rapid palatal expansion. Am J Orthod Dentofacial Orthop. 1998;114:686–696. 3. Stashenko P, Jandinski JJ, Fujiyoshi P, Rynar J, Socransky SS. Tissue levels of bone resorptive cytokines in periodontal disease. J Periodontol. 1991;62:504–509. 4. Schweikl SG, Hiller KA. Release of prostaglandin E2, IL-6 and IL-8 from human oral epithelial culture models after exAngle Orthodontist, Vol 75, No 4, 2005

636

5.

6.

7.

8.

9. 10.

11.

12.

13.

14. 15.

16.

17.

18.


posure to compounds of dental materials. Eur J Oral Sci. 2000;108:442–448. Sandy JR, Meghji S, Harris M. Bone remodeling in the jaws—clinical considerations. In: Harris M, Edgar M, Meghji S, eds. Clinical Oral Science. Oxford, UK: Reed Profess Publ Ltd; 1998:109–115. Adams DF. Occlusion and periodontal disease. In: Wentz FM, ed. Principles and Practice of Periodontics with an Atlas of Treatment. Springfield, Ill: Charles C Thomas Publ; 1978; 249–258. Vandevska RV, Kvinsland IH, Kvinsland S, Jonsson R. Immunocompotent cells in rat periodontal ligament and their recruitment incident to experimental orthodontic tooth movement. Eur J Oral Sci. 1997;105:36–44. Baggiolini M, Walz A, Kunkel SL. Neutrophil-activating peptide-1/IL-8, a novel cytokine that activates neutrophils. J Clin Invest. 1989;84:1045–1049. Shaw MM, Waters NE. The characteristics of the Ricketts maxillary canine retractor. Eur J Orthod. 1992;14:37–46. Chung RM, Grbic JT, Lamster IB. Interleukin-8 and b-glucuronidase in gingival crevicular fluid. J Clin Periodontol. 1997;24:146–152. Mathur A, Michalowicz B, Castillo M, Aeppli D. Interleukin1 alpha, interleukin-8 and interferon-alpha levels in gingival crevicular fluid. J Periodontal Res. 1996;31:489–495. Payne JB, Reinhardt RA, Masada MP, Dubois LM, Allison AC. Gingival crevicular fluid IL-8: correlation with local IL1b levels and estrogen status. J Periodontal Res. 1993;28: 451–453. Gianelly AA. Force induced changes in the vascularity of periodontal ligament. Am J Orthod Dentofacial Orthop. 1969; 55:5–15. Lanyon LE, Rubin CT. Static vs. dynamic loads as an influence on bone remodeling. J Biomech. 1984;17:897–905. Duncan GW, Yen E, Pritchard ET, Suga DM. Collagen and prostaglandin synthesis in force stressed periodontal ligament in vitro. J Dent Res. 1984;63:665–669. Hickory WB, Nanda R. Effect of tensile force magnitude on release of cranial suture cells into S phase. Am J Orthod Dentofacial Orthop. 1987;91:328–334. Meikle MC, Heath JK, Hembry RM, Reynolds JJ. Rabbit cranial suture fibroblasts under tension express a different collagen phenotype. Arch Oral Biol. 1982;27:609–613. Alhashimi N, Frithiof L, Brudvik P, Bakhiet M. Orthodontic tooth movement and de novo synthesis of proinflammatory

Angle Orthodontist, Vol 75, No 4, 2005

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

cytokines. Am J Orthod Dentofacial Orthop. 2001;119:301– 312. Griffiths GS, Moulson AM, Petrie A, James IT. Evaluation of osteocalcin and pyridinium crosslinks of bone collagen as markers of bone turnover in gingival crevicular fluid during different stages of orthodontic treatment. J Clin Periodontol. 1998;25:492–498. Ren Y, Maltha JC, Hof MA, Von den Hoff JW, Kuipers-Jagtman AM, Zhang D. Cytokine levels in gingival crevicular fluid are less responsive to orthodontic force in adults than in juveniles. J Clin Periodontol. 2002;29:752–762. Kurdowska AK, Noble JM, Adcock JE. Interleukin-8 and anti-interleukin-8 autoantibodies in gingival crevicular fluid from patients with periodontitis. J Periodontol. 2003;38:73– 78. ¨ zmeric¸ N, Bal B, Balos¸ K, Berker E, Bulut S¸. The correO lation of gingival crevicular fluid Interleukin-8 levels and periodontal status in localized juvenile periodontitis. J Periodontol. 1998;69:1299–1304. Takahashi I, Nishimura M, Onodera K, Bae JW, Mitani H, Okazaki M, Sasano Y. Expression of MMP-8 and MMP-13 genes in the periodontal ligament during tooth movement in rats. J Dent Res. 2003;82:646–651. Lee KJ, Park YC, Yu HS, Choi SH, Yoo YJ. Effects of continuous and interrupted orthodontic force on interleukin-1b and prostaglandin E2 production in gingival crevicular fluid. Am J Orthod Dentofacial Orthop. 2004;125:168–177. Ngan P, Saito S, Lanese R, Shanfeld J, Davidovitch Z. The interactive effects of mechanical stress and interleukin-1 on prostaglandin E and cycle AMP production in human periodontal ligament fibroblasts in vitro: comparison with cloned osteoblastic cells of mouse. Arch Oral Biol. 1990;35:717– 725. Saito S, Sait OM, Ngan P, Lanese R, Shanfeld J, Davidovitch Z. Effect of parathyroid hormone and cytokines on prostaglandin E synthesis and bone resorption by human periodontal ligament fibroblasts. Arch Oral Biol. 1990;35:845– 855. Iwasaki LR, Haack JE, Nickel JC, Reinhardt RA, Petro TM. Human interleukin-1b and interleukin-1 receptor antagonist secretion and velocity of tooth movement. Arch Oral Biol. 2001;46:185–189. Alhashimi N, Frithiof L, Brudvik P. Chemokines are upregulated during orthodontic treatment. J Interferon Cytokine Res. 1999;19:1047–1052.