Interleukin-1β Activity and Collagen Synthesis in Human Dental Pulp Fibroblasts

JOURNAL OF ENDODONTICS Copyright © 2002 by The American Association of Endodontists

Printed in U.S.A. VOL. 28, NO. 3, MARCH 2002

Interleukin-1␤ Activity and Collagen Synthesis in Human Dental Pulp Fibroblasts Rahmat A. Barkhordar, DMD, Q. Perveen Ghani, PhD, Thomas R. Russell, DDS, and M. Zamirul Hussain, PhD

of this cytokine in humans and seems to be important in the pathogenesis of chronic-pulpal inflammation. We hypothesize that IL-1␤ may be involved in accelerated collagen synthesis by chronically diseased pulps. The specific aims of this study were to determine the level of IL-1␤ in fibroblasts derived from healthy and chronically diseased pulps and to determine the effect of exogenous IL-1␤ on collagen synthesis in cultures of these fibroblasts.

Immunopathologic reactions play a significant role in inflammatory diseases of dental pulp. Interleukin-1␤ (IL-1␤) is recognized as a key player in mediating cellular immune response. In this study, we measured the content of IL-1␤ and its effect on collagen synthesis in cultures of fibroblasts derived from healthy and diseased dental pulps. We found that diseased pulp fibroblasts contain 2.5fold greater amounts of IL-1␤ and synthesized 80% greater amounts of collagen compared with healthy pulp fibroblasts. However, exogenous IL-1␤ failed to stimulate collagen synthesis by diseased fibroblasts, whereas collagen synthesis by healthy pulp fibroblasts was stimulated by more than 2-fold. These observations imply that pulp disease induces abnormalities associated with fibroblast response toward IL-1␤.

MATERIALS AND METHODS Collection of Pulp Tissues Diseased pulp tissues were obtained from teeth diagnosed with pulpitis. Normal tissue was from freshly extracted impacted third molars, which were used as a control. Freshly extracted teeth were placed in liquid nitrogen and carried to the laboratory for sectioning. Two longitudinal grooves were made on each side of the tooth with a diamond disk. With a surgical chisel, the teeth were cracked open, and the pulp tissue was removed with a cotton plier. For growing fibroblasts, tissues were immersed in cell culture medium that was maintained at 37°C.

The dental pulp is contained in a protective wall of hard tissue. Once the integrity of this outer shield is lost, the pulp is exposed to mechanical and bacterial insults and subject to inflammation. Pulpal inflammation in response to caries begins as a low-grade, chronic response that lacks an acute phase (1). Although inflammation leads to repair of other injured tissues, the diseased pulp in a state of chronic inflammation usually does not heal. Many etiologic factors, such as fractures, caries, and restorative procedures, are known to cause pulpal inflammation, but the pathogenesis of chronic-pulpal inflammation remains unclear. Chronic-pulp inflammation has been characterized by the presence of macrophages, lymphocytes, and plasma cells (2). With time, cellular elements decrease, and the collagen content of the pulp increases (3). This collagen may become a site for calcification, which leads to the production of pulp stones and other pulpal calcifications. Interleukin (IL)-1 has been identified in the gingival fluid of inflammatory periodontitis patients (4) and in pulpal periapical disease (5). D’Souza et al. (6) used a bioassay to show that pulps from carious teeth had higher levels of IL-1 and used immunohistochemistry to localize the cytokine to pulpal macrophages within the connective-tissue stroma. IL-1␤ is the predominate active form

Fibroblasts Cultures Pulp tissues were minced into 1 ⫻ 1-mm pieces with surgical scissors immersed in Dulbecco’s Eagle’s modified medium (DMEH16); fibroblasts were grown from tissue pieces in DME-H16 that contained 10% newborn calf serum supplemented with a mixture of antibiotics (60 ␮g/ml of penicillin G; 100 ␮g/ml of streptomycin; 76 ␮g/ml of neomycin; 2.5 ␮g/ml of amphotericin; and 300 ␮g/ml of L-glutamine. Cells were maintained in a humidified 5% CO2/95% air incubator at 37°C (7). The cultures were grown to confluence in 100-mm dishes (Costar Corp., Cambridge, MA). Cells in passages 4 through 6 were used for biochemical experiments. For cytokine treatment, cells were serum starved for 48 h, and quiescent fibroblasts were then treated with IL-1␤ for 20 h. To estimate cell numbers, an aliquot of trypsin (0.2%) was added to an experimental plate, after which an aliquot of the cell suspension was combined with a dispersing solution in a 1:20 ratio and counted in a Coulter counter. 157

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TABLE 1. Interleukin-1␤ in cultures of pulp fibroblasts Fibroblasts

IL-␤/mg protein (picograms)

Healthy pulp Healthy gingiva Diseased pulp

754 ⫾ 152 1152 ⫾ 130* 1898 ⫾ 201*

Cultures were prepared from pulp tissues, and IL-1␤ was estimated in the supernatant of cell homogenate as described in Materials and Methods. Data represent mean ⫾ SD from four culture dishes. * Value is significantly different from cultures from healthy pulp (p ⬍ 0.01, ANOVA).

Assay for IL-1␤ IL-1␤ was determined by the procedure that was described earlier by using the Quantikine IL-1␤ ELISA kit (5). Briefly, 100 ␮l of each standard (reconstituted with 0.5 ml distilled water) and test samples, containing 50 to 100 ␮g of protein, were placed in wells of microtitration plates and were incubated at 37°C for 2 h. The wells were emptied and washed three times with 250 ␮l of wash buffer. After the wash, 100 ␮l of IL-1 monoclonal antibody was added to each well and were incubated at 37°C for 2 h. The wells were washed again three times; 100 ␮l of antirabbit IgG/ horseradish peroxidase conjugate was then added, and the plate was incubated for 30 min at room temperature. After the wash, 100 ␮l of freshly prepared O-phenylenediamine substrate (one tablet dissolved in 5 ml of distilled water) was added to each well, and the plate was incubated at room temperature for 15 min. Finally, 50 ␮l of 4 N sulfuric acid was added to each well, and the color intensity was measured in a microtitration plate reader set at 490 nm. The amount of IL-1␤ in each sample was determined from the standard curve and was expressed in picograms per milligram of protein, which was assayed by the method of Bradford by using Biorad dye reagent (8). Blank values were obtained with glassdistilled water and all other additions. Synthesis of Protein and Collagen Collagen and total protein productions were determined in conditioned media and cell layers of cultures labeled with 5 ␮Ci/ml of L-[2,3,4,5-3H]proline (73 Ci/nmol; Amersham, Piscataway, NJ) plus 50 ␮g/ml ascorbic acid in the presence (10 –50 pg/ml) and absence of IL-1␤ in DME-H16 for 20 h (7). At the end of the incubation, the medium was aspirated. Cells were scraped and homogenized in 0.1 M Tris-HCl, pH 7.5, containing 0.01% Triton X-100. Both the cell homogenate and the conditioned medium were dialyzed to remove free [3H] proline. Total protein synthesis was evaluated by measuring undialyzable radioactivity. The incorporation of [3H] proline into collagen was determined in the undialyzable-radiolabeled fraction by measuring the release of radioactivity in dialyzable peptides upon digestion with purified Clostridium collagenase (Advanced Biofractures Corp., Lynbrook, NY) at 37°C for 8 h (9). RESULTS Table 1 shows the levels of IL-1␤ in cultures of fibroblasts grown from healthy and diseased pulp tissues. Gingival fibroblasts were used as a positive control. All samples displayed measurable amounts of cytokine. However, the cultures from diseased pulp showed nearly 3-fold greater amounts of IL-1␤ compared with those from healthy pulp.

TABLE 2. Effect of interleukin-1␤ on protein and collagen synthesis by fibroblasts derived from healthy and diseased pulp H-proline Incorporation (dpm ⫻ 104/106 cells)

CollagenaseSensitive Radioactivity (dpm ⫻ 104/106 cells)

12.73 ⫾ 1.7

1.09 ⫾ 0.12

18.41 ⫾ 2.0* 17.98 ⫾ 2.0* 11.77 ⫾ 2.0

2.21 ⫾ 0.28* 2.33 ⫾ 0.25* 1.14 ⫾ 0.13

18.53 ⫾ 2.4* 19.21 ⫾ 2.3*

2.35 ⫾ 0.20* 2.52 ⫾ 0.26*

16.63 ⫾ 2.0

1.83 ⫾ 0.22

15.85 ⫾ 1.9 15.52 ⫾ 1.7 19.01 ⫾ 2.5

1.63 ⫾ 0.23 1.71 ⫾ 0.21 1.87 ⫾ 0.24

16.23 ⫾ 2.1 15.97 ⫾ 2.5

1.66 ⫾ 0.21 1.81 ⫾ 0.19

20.76 ⫾ 2.3

1.62 ⫾ 0.21

26.13 ⫾ 3.1

2.61 ⫾ 0.35*

3

Treatment

Healthy 1, untreated IL-1, 50 pg/ml 100 pg/ml Healthy 2, untreated IL-1, 50 pg/ml 100 pg/ml Diseased 1, untreated IL-1, 50 pg/ml 100 pg/ml Diseased 2, untreated IL-1, 50 pg/ml 100 pg/ml Healthy gingival, untreated IL-1, 50 pg/ml

Synthesis of total protein and collagen were determined in cultures as described in Materials and Methods. Each value is mean ⫾ SD of data from three independent determinations. Gingival fibroblasts were used as control. * Value is significantly different from untreated cultures (p ⬍ 0.01, ANOVA).

Table 2 shows the synthesis of total protein and collagen by cultures of fibroblasts exposed to the recombinant IL-1␤. Fibroblasts derived from two healthy and two diseased pulp tissues maintained in the same passage were used for this experiment. Cultures from healthy pulp synthesized twice the amount of extracellular collagen in the presence of the cytokine. Collagen stimulation was more pronounced than the increase in total protein. Gingival fibroblasts, which were used as control, exhibited similar collagen stimulation. On the other hand, the cultures derived from diseased pulp behaved quite differently. These cultures synthesized 80% greater amounts of collagen compared with fibroblasts from healthy pulp. However, no significant effect of IL-1␤ was evident. Cultures from diseased pulp produced similar amounts of extracellular collagen in the presence and absence of added IL-1␤ (Table 2 and Fig. 1). Figure 1 displays fibroblast response from independent cultures derived from two patients. DISCUSSION The data from this study show that IL-1␤ is present in diseased pulpal-fibroblast cell cultures in significantly greater amounts than in healthy pulpal-fibroblast cell cultures. Hosoya and Matsushima (10) showed that IL-1␤ release and mRNA levels in human dental pulpal cells were increased by lipopolysaccharides from Porphyromonas endodontalis. Thus, IL-1␤ seems to be involved in the pulpal-inflammatory response to bacterial invasion. The incorporation of tritiated-proline data seems to show a marked difference between healthy pulpal fibroblasts and fibroblasts of pulps of carious teeth with respect to their levels of collagen synthesis. Our results seem to show that diseased fibro-

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dition of IL-1␤. Apparently, the diseased pulpal cells are already turned on for collagen synthesis and are unable to respond to exogenous IL-1␤. We propose that diseased pulpal fibroblasts are altered in some intracellular mechanism that is associated with the cells’ ability to stimulate collagen synthesis in the presence of effector cytokine. Prostaglandins may be involved, or alternatively, high levels of IL-1␤ receptor antagonist may be present, which would competitively inhibit the effect of adding IL-1␤ (17). Further study is warranted to elucidate the regulation of collagen synthesis in diseased fibroblasts. Therapeutic value might become evident if it were possible to retard the process of fibrosis and calcification in response to caries and restorative procedures. FIG. 1. Collagen synthesis by pulpal and gingival fibroblasts in the presence of interleukin-1␤. Data for collagen synthesis are taken from Table 2. Values represent an average of two separate cultures, derived from two patients. Each number represents mean ⫾ SD from three culture dishes. *Value is significantly different from untreated cultures (p ⬍ 0.01, ANOVA).

blasts have a significantly higher level of collagen synthesis, which would correspond to a chronically inflamed pulp becoming more fibrotic. This fibrotic material may then become a site for increased formation of pulpal calcifications. Our findings, with regard to IL-1␤ mediation of collagen synthesis, agree with those of Lertchirakarn et al. (11), who found a mild stimulation of Type I collagen synthesis by human-pulpal fibroblasts in response to IL-1␤ in both the presence and absence of indomethacin. Goldring and Krane (12) also showed that stimulation of collagen synthesis by IL-1␤ treatment of synovial fibroblasts and chondrocytes was unmasked by inhibition of prostaglandin E2 synthesis by indomethacin. Preincubation with IL-1␤ alone decreased synthesis of Type I collagen. Postlethwaite et al. (13) found that human recombinant IL-1␤ stimulated fibroblasts to increase procollagen gene expression at the pretranslational level and that indomethacin had no measurable effect on the ability of IL-1 to stimulate production of collagen. Diaz et al. (14) found that in lung fibroblasts, IL-1␤ inhibits the expression of the alpha (I) procollagen gene at the transcriptional level by a PGE2-independent effect and also stimulates release of endogenous-fibroblast PGE2, leading to inhibition of procollagen production. Rapala et al. (15) treated experimental granulation tissue in rats and found that IL-1␤ decreased collagen production by 15% and that this effect could be abolished by indomethacin, which alone stimulated collagen production by 40%. It may be that fibroblasts of different origins respond differently to IL-1␤ than do human-pulpal fibroblasts. Inflammation generally induces greater production of cytokines and growth factors by many cells (16). In this study, we found significantly elevated levels of IL-1␤ induced in fibroblasts that were isolated from diseased pulp. This may have caused the stimulation in collagen synthesis, compared with healthy cells, which are normally exposed to lower levels of IL-1␤. Cultures derived from both healthy gingiva and healthy pulp responded similarly to the exogenous IL-1␤ and increased collagen production. This suggests that cytokine stimulation of collagen is a normal cellular response (12, 13). Addition of IL-1␤ to healthy human-pulpal fibroblasts, in this study, stimulated them to double their collagen synthesis. This is in contrast to the result seen with diseased fibroblasts, in which no significant difference in collagen synthesis was evident upon ad-

Drs. Barkhordar, Ghani, Russell, and Hussain are affiliated with the Department of Preventive & Restorative Dental Sciences, School of Dentistry, University of California, San Francisco, CA. Address requests for reprints to Dr. Rahmat A. Barkhordar, Associate Professor, Division of Endodontics, School of Dentistry, University of California, San Francisco, CA 94143.

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