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Effects of interferons on proliferation and collagen synthesis of rat palatal wound fibroblasts
Archives of Oral Biology 44 (1999) 541±547
Eects of interferons on proliferation and collagen synthesis of rat palatal wound ®broblasts A.M.H. Cornelissen*, J.W. Von den Ho, J.C. Maltha, A.M. KuijpersJagtman Department of Orthodontics and Oral Biology, College of Dental Sciences, Medical Faculty, University of Nijmegen, P.O. Box 9101, NL-6500 HB, Nijmegen, The Netherlands Accepted 23 February 1999
Abstract The purpose was to select drugs that speci®cally reduce collagen synthesis by palatal granulation ®broblasts without aecting their proliferation. Granulation ®broblasts were obtained from 8-day-old palatal mucoperiosteal wounds and normal ®broblasts from palatal tissue of unwounded rats. Cultured cells were treated with interferona2b, interferon-b and interferon-g (0, 100, 1000, and 10000 U/ml). Cell proliferation was measured by [3H]thymidine incorporation. Collagen synthesis and non-collagenous protein synthesis were determined from the incorporation of [3H]proline. None of the interferons signi®cantly inhibited the proliferation of either type of ®broblasts. Interferona2b had no eect on the variables studied at the dosages used. Interferon-b reduced collagen synthesis of granulation ®broblasts without aecting their non-collagenous protein synthesis or protein synthesis by normal ®broblasts. Interferon-g reduced collagen synthesis of both types of ®broblast and the non-collagenous protein synthesis of granulation ®broblasts. These data show that interferon-b speci®cally reduces collagen synthesis by oral granulation ®broblasts without aecting normal palatal ®broblasts. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Interferon; Wound healing; Oral ®broblasts; Collagen synthesis
1. Introduction Scar formation after cleft palate surgery is one of the major factors causing aberrant maxillary growth and dento-alveolar development (Ross, 1987). Consequently, the inhibition of scar formation seems a feasible way of improving the clinical outcome. Tissue injury triggers a complex cascade of cellular and biochemical events that ®nally results in a scar.
Abbreviations: DMEM, Dulbecco's modi®ed Eagle's medium; FCS, fetal calf serum; IFN, interferon; NEM, N-ethylmaleimide; TCA, trichloroacetic acid. * Corresponding author. Fax: +00-31-243-540631.
After injury, there is in¯ammation, ®broblast proliferation, matrix synthesis and remodelling (Witte and Barbul, 1997). Every phase contributes to the amount of scarring by in¯uencing the rate, quality and total amount of matrix deposition. Oral palatal wounds also pass through these phases of wound healing (Kahnberg and Thilander, 1982). The number of collagen ®bres in palatal mucoperiosteal wounds is increased up to 4 weeks after wounding (Searls et al., 1979). This seems to be due not only to an increased number of cells but also to an increase in collagen production per cell (Moriyama et al., 1991). A comparison of adult and foetal wound healing has shown that foetal wounds heal without scarring (Longaker and Adzick, 1991). This has been attributed
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to a lower in¯ammatory response (Whitby and Ferguson, 1991a) and a dierent cytokine pro®le (Whitby and Ferguson, 1991b), which suggests that manipulation of wound healing by altering the cytokine pro®le could lead to scarless healing. Only a few cytokines have been identi®ed that have the potential to reduce scarring; interferons represent the ®rst wellde®ned group of polypeptides that exert this property (Granstein et al., 1990a). The ability of interferons to modulate wound healing has been studied in vivo and on a variety of cell types in vitro. IFN-a, IFN-b and IFN-g reduce collagen synthesis by dermal ®broblasts (Duncan et al., 1995), hypertrophic scar ®broblasts (Tredget et al., 1993) and ®broblasts derived from sclerodermal lesions (Duncan and Berman, 1987). In vivo studies in mice have demonstrated that IFN-g reduced the increased collagen synthesis associated with the ®brotic response to an implanted foreign body (Granstein et al., 1990a). This eect was also found in bleomycin-induced pulmonary ®brosis and the healing response to cutaneous burn wounds (Granstein et al., 1990a). The therapeutic relevance of these in vitro and in vivo observations has been con®rmed in clinical trials. Intralesional injection of IFN-a2b (Berman and Duncan, 1989) or IFN-g (Granstein et al., 1990b) reduced the sizes of keloid and hypertrophic scars (Larrabee et al., 1990). The regulation of protein and collagen synthesis in normal ®broblasts by interferon in vitro depends on the anatomical site (Smith and Higgins, 1993; Martens et al., 1992). Moreover, dermal ®broblasts show a modulated response to IFN-g according to their stage of dierentiation during wound healing (Moulin et al., 1998). For wound healing factors such as migration (Irwin et al., 1994) and matrix remodelling (Stephens et al., 1996), it has been shown that there are phenotypic dierences between dermal and oral ®broblasts. Therefore it is quite possible that there are dierences in the regulation of proliferation and protein synthesis by interferons between dermal and oral ®broblasts, and between ®broblasts from palatal granulation tissue and normal palatal tissue. Our aim now was to determine the eects of interferons on proliferation, collagen synthesis, and noncollagenous protein synthesis of mucoperiosteal granulation and normal ®broblasts. 2. Materials and methods 2.1. Interferons Human recombinant IFN-a2b was obtained from Schering Plough BV (Amstelveen, The Netherlands). Fibroblast-derived human IFN-b was obtained from ICN Pharmaceuticals (Costa Mesa, USA) and rat
recombinant IFN-g was kindly provided by Dr P.H. van der Meide, (BPRC Rijswijk, The Netherlands). 2.2. Fibroblast cultures Five-week-old male Wistar rats were anaesthetized with a subcutaneous injection of 0.4 ml/kg body wt fentanyl+¯uanisone (Hypnorm1; Janssen-Cilaq, Beerse, Belgium). A standardized wound was made in the mucoperiosteum of the hard palate between the second molars using a 3-mm biopsy punch. The animals were medicated postoperatively with 0.1 mg/kg body wt buprenor®ne (Temgesic1; Schering Plough BV) as an analgesic. After 8 days, the rats were decapitated. Punch biopsy samples of the granulation tissue were collected in DMEM supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml fungizone, and 10% FCS, termed culture medium. Normal ®broblasts were obtained from biopsy samples of palatal mucosa of age-matched control rats. The samples of granulation tissue and normal mucoperiosteum were washed twice with culture medium and minced into small pieces. Primary ®broblast cultures were established from these explants. The cultures were maintained at 378C, 5% CO2 in culture medium in a humidi®ed incubator. Fibroblasts were passaged at con¯uence by trypsinization. All experiments were done on cells at low passage (<5). All culture supplies were purchased from Life Technologies (Breda, The Netherlands). 2.3. Proliferation assay Trypsinized ®broblasts were plated in 96-well microtitre plates at a density of 2000 cells/well in 100 ml culture medium. After 24 h, the cells were synchronized with culture medium containing 0.5% FCS for 24 h. Subsequently, the medium was replaced by 100 ml culture medium for 24 h. Then the medium was removed and replaced with 100 ml culture medium containing 420 kBq/ml [methyl-3H]thymidine (Amersham, Little Chalfont, UK) and various concentrations of IFNa2b, IFN-b, or IFN-g (0, 100, 1000, and 10000 U/ml). After 19 h, the medium was removed and the cells were washed three times with DMEM. Finally, the cells were harvested on a glass-®bre ®lter (LKBWallac, Turku, Finland) using a cell harvester (LKBWallac) and the incorporation of [methyl-3H]thymidine into DNA was counted with a betaplate liquid scintillation counter (LKB-Wallac). 2.4. Collagen and non-collagenous protein synthesis assay Collagen production by con¯uent monolayers was determined by the incorporation of [5-3H]proline into
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Table 1 Collagen and non-collagenous protein synthesis by palatal mucoperiosteal granulation tissue ®broblasts (GF) and normal palatal ®broblasts (NF)a
Collagen synthesis Non-collagenous protein synthesis a
counts/min per ng DNA counts/min per ng DNA
GF
NF
p
481.7250 821.3265
338.8244 660.8271
0.04 0.11
Data represent the mean value2SE of nine experiments.
bacterial collagenase-sensitive protein. The method described previously by Martens et al. (1992) was modi®ed to measure collagen synthesis in small numbers of ®broblasts. Trypsinized ®broblasts were dispensed into 24-well plates at a density of 20,000 cells/ well in 400 ml of medium and were grown until con¯uence. The medium was replaced with 400 ml of medium containing 50 mg/ml L-ascorbic acid (Sigma, St. Louis, USA), 164 kBq/ml L-[3H]proline (Amersham), and various concentrations of IFN-a2b, IFN-b, or IFN-g (0, 100, 1000, and 10000 U/ml). After the labelling period, cells and medium were collected and washed as described by Martens et al. (1992). The precipitate of cells and medium was dissolved in 550 ml 0.2 M NaOH at room temperature and neutralized with 445 ml 0.15 M HCl and 525 ml 1 M HEPES, pH 7.2 (Life Technologies). The solution was divided into three portions. (1) To determine the incorporation into total proteins, 200 ml was placed in a scintillation vial. (2) To determine the incorporation into collagen, 400 ml was placed in a 2-ml vial and digested by collagenase. (3) Another 400 ml was also placed in a 2-ml vial as a blank for the second portion. Collagenase digestion was performed by adding 400 ml highly puri®ed collagenase (ICN; 15 BTC units/ml) dissolved in 100 mM CaCl2H2O, 0.1 M TRIS and 2 mM NEM, pH 7.2. The blank digestion was done without collagenase. The second and third portions were incubated for 3 h at 378C. They were treated with 400 ml 15% TCA and 0.1% tannic acid (Sigma), centrifuged at 5000 g for 7 min, and the supernatant was placed in a scintillation vial. The pellet was resuspended and washed with 500 ml 5% TCA and 0.1% tannic acid. Aqua Luma (Lumac-LSC, Groningen, The Netherlands) was added as a scintillator, and the samples were measured by liquid scintillation counting (LKB-Wallac). Unless stated otherwise, the chemicals were obtained from Merck (Darmstad, Germany). The speci®city of this method was tested by labelling with [3H]tryptophan instead of [3H]proline, as trytophan is not present in collagen. The purity of the collagen fraction was 99.3%. The collagen synthesis was determined by subtraction of the counts released into the blank incubation
from those released in the presence of collagenase. The non-collagenous protein synthesis was calculated by subtraction of the radioactivity of the collagen synthesis from that of total protein. Collagen synthesis and non-collagenous protein synthesis were corrected for the amount of DNA per well with the PicoGreen1 dsDNA quantitation kit (Molecular Probes, Eugene, USA). 2.5. Statistical analysis Eighteen rats were used in total. From explants of nine wounded and nine control rats, nine separate cultures of granulation ®broblasts and nine separate cultures of normal ®broblasts were established. Three cultures of each type (granulation, normal) were used for every type of interferon (IFN-a2b, -b, -g). Each type of interferon was tested in triplicate cultures at a concentration of 0, 100, 1000, and 10000 U/ml. Values are expressed as the mean value relative to untreated cultures2SE. The collagen and non-collagenous protein synthesis of granulation ®broblasts was compared with that of normal ®broblasts by the student's t-test. The eects of the dierent concentrations of interferon (100, 1000 and 10000 U/ml) were compared with the control values (0 U/ml) by two-way analysis of variance on repeated measures. This was done in order to consider variations between ®broblasts from one rat to another. Signi®cant dierences were further analysed by Tukey's multiple-comparisons test. A p-value of less than 0.05 was considered to be statistically signi®cant. 3. Results 3.1. Collagen and non-collagenous protein synthesis of untreated ®broblasts Palatal granulation ®broblasts produced signi®cantly 42% more collagen than normal ®broblasts. Although not signi®cant, granulation ®broblasts produced 24% more non-collagenous proteins than normal ®broblasts (Table 1).
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Fig. 1. The eects of IFN-a2b on proliferation (A), collagen synthesis (B), and non-collagenous protein synthesis (C) of ®broblasts. Black bars represent palatal granulation-tissue ®broblasts and white bars normal ®broblasts. The results are expressed as percentage of untreated control cultures. Values are means2SE of three experiments. =p < 0.05.
Fig. 2. The eects of IFN-b on proliferation (A), collagen synthesis (B), and non-collagenous protein synthesis (C) of ®broblast. Black bars represent palatal granulation-tissue ®broblasts and white bars normal ®broblasts. The results are expressed as percentage of untreated control cultures. Values are means2SE of three experiments. =p < 0.05.
3.2. Interferon-a2b
collagenous protein synthesis in granulation ®broblasts decreased only by 624% and 1525% in the presence of 1000 and 10000 U/ml, respectively (Fig. 2C), which was not statistically signi®cant.
Although IFN-a2b tended to reduce the proliferation (Fig. 1A) of granulation and normal ®broblasts by about 10±20%, there were no signi®cant eects. Also, the collagen (Fig. 1B) and non-collagenous protein synthesis (Fig. 1C) of granulation and normal ®broblasts were not signi®cantly aected after incubation with IFN-a2b for 24 h. 3.3. Interferon-b IFN-b had no signi®cant eect on the proliferation of granulation or normal ®broblasts (Fig. 2A). Addition of IFN-b to cultures of normal ®broblasts did not aect collagen synthesis. By contrast, collagen synthesis by granulation ®broblasts was signi®cantly decreased, by 37213% and 3429% in the presence of 1000 and 10000 U/ml, respectively (Fig. 2B). The non-
3.4. Interferon-g IFN-g did not signi®cantly reduce the proliferation of granulation and normal ®broblasts (Fig. 3A). Collagen synthesis by granulation ®broblasts was signi®cantly reduced by 4727, 4326 and 4227% in the presence of 100, 1000, and 10000 U/ml, respectively (Fig. 3B). The suppression of collagen synthesis was not cell-type speci®c as synthesis by normal ®broblasts was also reduced by 31 2 4, 36 2 4, 31 2 8%, respectively. The eects of IFN-g on non-collagenous protein synthesis were more pronounced in granulation than in normal ®broblasts (Fig. 3C). A non-signi®cant suppression was found in normal ®broblasts, whereas a
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Fig. 3. The eects of IFN-g on proliferation (A), collagen synthesis (B), and non-collagenous protein synthesis (C) of ®broblasts. Black bars represent palatal granulation-tissue ®broblasts and white bars normal ®broblasts. The results are expressed as percentage of untreated control cultures. Values are means2SE of three experiments. =p < 0.05.
signi®cant decrease occurred in granulation ®broblasts by 3523, 3224 and 2727% in the presence of 100, 1000 and 10000 U/ml, respectively. 4. Discussion We have determined the eects of interferons on proliferation, collagen and non-collagenous protein synthesis of granulation-tissue ®broblasts. We have evidence that in this rat model the vast majority of ®broblasts in the granulation tissue are myo®broblasts at 8 days after wounding (A.M.H. Cornelissen et al., unpublished data). Myo®broblasts are involved in wound contraction, matrix synthesis, and remodelling. They have been associated with scar tissue formation (Estes et al., 1994), hypertrophic scarring and ®bromatosis (DesmoulieÁre and Gabbiani, 1996). Therefore we used cells derived from 8-day-old palatal mucoperios-
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teal wounds. The cells were not frozen and the number of passages was never more than ®ve, in order to preserve their original biosynthetic properties as much as possible. Fibroblast metabolism is modi®ed after wounding by other cells, the extracelllular matrix, and cytokines leading, for example, to an increased matrix synthesis (Mutsaers et al., 1997). We have found that rat palatal granulation ®broblasts cultured from 8-day-old wounds synthesize 42% more collagen than normal ®broblasts. In the same culture model, others have found that immature scar ®broblasts of 1-month-old palatal wounds produce 60% more collagen than normal palatal rat ®broblasts (Moriyama et al., 1991). Apparently, collagen production by mucoperiostealwound ®broblasts is upregulated for longer. It has recently been reported that human granulation ®broblasts taken from 12-day-old dermal wounds synthesize ®ve times more collagen than normal ®broblasts (Moulin et al., 1998). These ®ndings and our present results suggest that collagen synthesis by dermal ®broblasts after wounding is more upregulated than that of palatal ®broblasts. IFN-a, -b, and -g decrease the proliferation and collagen synthesis of ®broblasts derived from both normal and ®brotic skin (Duncan and Berman, 1985; Berman and Duncan, 1989; Tredget et al., 1993). The aim of our study was to compare the short-term eects of these interferons on proliferation, collagen and noncollagenous protein synthesis of palatal mucoperiosteal granulation or normal ®broblasts. We show that short-term exposure to IFN-a2b did not signi®cantly reduce the proliferation of palatal ®broblasts. A similar eect has been found in normal dermal ®broblasts (Duncan and Berman, 1985). Also, collagen synthesis by mucoperiosteal ®broblasts was not aected by IFN-a2b, which is in contrast with the suppressive eects of IFN-a2b on collagen synthesis by dermal ®broblasts (Duncan and Berman, 1985). This discrepancy may arise from dierences in type of ®broblasts but also from dierences in culture conditions. Although it is known that IFN-a stimulates immediate-response genes (Darnell et al., 1994), there are strong indications that it suppresses collagen synthesis by an indirect mechanism that takes approx. 48 h to have a signi®cant eect (Tredget et al., 1993). Therefore, it is possible that long-term exposure to IFN-a2b can reduce collagen synthesis by palatal ®broblasts. IFN-b had no eect on the proliferation of granulation or normal ®broblasts. It reduced collagen synthesis by granulation ®broblasts by about 35% and did not aect normal ®broblasts. It can be concluded that granulation ®broblasts dier from normal ®broblasts in their response to IFN-b. On the other hand, non-collagenous protein synthesis was not reduced by
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IFN-b in granulation and normal ®broblasts. These dierences in response to IFN-b between palatal granulation and normal ®broblasts are probably due to an intrinsic dierence as both cell types were treated in the same way. A dierential response of palatal scar ®broblasts and normal ®broblasts to cytokines such as transforming growth factor-b1, epidermal growth factor and platelet-derived growth factor has already been reported (Moriyama et al., 1991). IFN-g is generally known as an inhibitor of cell growth. We have found that it does not signi®cantly suppress the proliferation of palatal granulation or normal ®broblasts. Others reported that IFN-g stimulates the growth of human synovial ®broblasts and dental pulp ®broblasts (Butler et al., 1988; Melin et al, 1989). This discrepancy may arise from dierences in the type of ®broblasts but also from dierences in experimental conditions, such as the duration of treatment. Numerous studies have shown that IFN-g inhibits collagen synthesis by dermal ®broblasts, ®broblasts taken from ®brotic lesions (Granstein et al., 1990a) and dermal granulation ®broblasts (Moulin et al., 1998). In this respect, palatal normal and granulation ®broblasts behave the same. IFN-g regulates collagen synthesis by reducing the transcription rate of the COL1A1 and COL3A1 gene, the stability of the corresponding transcript and the rate of translation (Diaz and Jimenez, 1997). In contrast to IFN-b, IFN-g suppressed collagen synthesis by palatal granulation ®broblasts non-speci®cally, as non-collagenous protein synthesis was also reduced. Others have found that IFN-g does not aect non-collagenous protein synthesis of dermal granulation ®broblasts (Moulin et al., 1998). This discrepancy indicates that palatal granulation ®broblasts are perhaps more sensitive to IFN-g than dermal granulation ®broblasts. However, the dierence could also be related to dierences in culture conditions. Variations in potency to inhibit collagen synthesis between the interferons might be explained by the mechanisms of signal transduction. IFN-g (type II) receptors on ®broblasts have been demonstrated to be dierent from the type I interferon receptors. Although IFN-a and IFN-b both bind to the type I receptor (Darnell et al., 1994), they transduce dierent signals (Platanias et al., 1996) and can induce dierent genes (Sandhya Rani et al., 1996). In conclusion, IFN-b and IFN-g are both able to reduce collagen synthesis by palatal granulation ®broblasts in vitro. An optimal therapy against palatal scarring reduces the collagen synthesis of ®broblasts without aecting the synthesis of other proteins and without aecting normal ®broblasts. This means that IFN-b is the more appropriate interferon for further studies on scar reduction in vivo.
Acknowledgements We are grateful to Dr PH van der Meide (Biomedical Primate Research Centre, Rijswijk, The Netherlands) for the gift of IFN-g. References Berman, B., Duncan, M.R., 1989. Short-term keloid treatment in vivo with human interferon alfa-2b results in a selective and persistent normalization of keloidal ®broblast collagen, glycosaminoglycan, and collagenase production in vitro. Journal of the American Academy of Dermatology 21, 694±702. Butler, D.M., Piccoli, D.S., Hart, P.H., Hamilton, J.A., 1988. Stimulation of human synovial ®broblasts DNA synthesis by recombinant human cytokines. Journal of Rheumatology 15, 1463±1470. Darnell, J.E., Kerr, I.M., Stark, G.R., 1994. Jak-STAT Pathways and transcriptional activation in response to IFNs and other extracellular signalling proteins. Science 264, 1415±1421. DesmoulieÁre, A., Gabbiani, G., 1996. The role of the myo®broblasts in wound healing and ®brocontractive diseases. In: Clark, R.A.F. (Ed.), The molecular and cellular biology of wound repair. Plenum Press, New York, USA, pp. 391±423. Diaz, A., Jimenez, S.A., 1997. Interferon gamma regulates collagen and ®bronectin gene expression by transcriptional and post-transcriptional mechanisms. International Journal of Biochemistry and Cell Biology 29, 251±260. Duncan, M.R., Berman, B., 1985. g Interferon is the lymphokine and b interferon the monokine responsible for inhibition of ®broblast collagen production and late but not early ®broblast proliferation. Journal of Experimental Medicine 162, 516±527. Duncan, M.R., Berman, B., 1987. Persistence of reduced-collagen-producing phenotype in cultured scleroderma ®broblasts after short-term exposure to interferons. Journal of Clinical Investigation 79, 1318±1324. Duncan, M.R., Hasan, A., Berman, B., 1995. Pentoxifylline, pentifylline, and interferons decrease type I and III procollagen mRNA levels in dermal ®broblasts: evidence for mediation by nuclear factor 1 down regulation. Journal of Investigative Dermatology 104, 283±286. Estes, J.M., Vande-Berg, J.S., Adzick, N.S., MacGillvray, T.E., DesmoulieÁre, A., Gabbiani, G., 1994. Phenotypic and functional features of myo®broblasts in sheep wounds. Dierentiation 56, 173±181. Granstein, R.D., Flotte, T.J., Amento, E.P., 1990a. Interferons and collagen production. Journal of Investigative Dermatology 95, 75S±80S. Granstein, R.D., Rook, A., Flotte, T.J., Haas, A., Gallo, R.L., Jae, H.S., Amento, E.P., 1990b. A controlled trial of intralesional recombinant interferon-gamma in the treatment of keloid scarring. Clinical and histologic ®ndings. Archives of Dermatology 126, 1295±1301. Irwin, C.R., Picardo, M., Ellis, I., Sloan, P., Grey, A., McGurk, M., Schor, S.L., 1994. Inter-and intra-site het-
A.M.H. Cornelissen et al. / Archives of Oral Biology 44 (1999) 541±547 erogenity in the expression of fetal-like phenotypic characteristics by gingival ®broblasts: potential signi®cance for wound healing. Journal of Cell Science 107, 1333±1346. Kahnberg, K., Thilander, H., 1982. Healing of experimental excisional wounds in the rat palate. Histological study of the interphase in wound healing after sharp dissection. International Journal of Oral Surgery 11, 44±51. Larrabee, W.F., East, C.A., Jae, H.S., Stephenson, C., Peterson, K.E., 1990. Intralesional interferon gamma treatment for keloids and hypertrophic scars. Archives of Otolaryngology: Head & Neck Surgery 116, 1159±1162. Longaker, M.T., Adzick, N.S., 1991. The biology of fetal wound healing: a review. Plastic and Reconstructive Surgery 87, 788±798. Martens, M.F., Huyben, C.M., Hendriks, T., 1992. Collagen synthesis in ®broblasts from human colon: regulatory aspects and dierences with skin ®broblasts. Gut 33, 1664± 1670. Melin, M.D.J., Hartmann, D.J., Magloire, H., Falco, E., Auriault, C., Grimaud, J.A., 1989. Human recombinant gamma-interferon stimulates proliferation and inhibits collagen and ®bronectin production by human dental pulp ®broblasts. Cellular and Molecular Biology 35, 97±110. Moriyama, K., Shimokawa, H., Susami, T., Sasaki, S., Kuroda, T., 1991. Eects of growth factors on mucosal scar ®broblasts in cultureÐa possible role of growth factors in scar formation. Matrix 11, 190±196. Moulin, V., Castilloux, G., Auger, F.A., Garrel, D., O'Connor-McCourt, M.D., Germain, L., 1998. Modulated response to cytokines of human wound healing myo®broblasts compared to dermal ®broblasts. Experimental Cell Research 238, 283±293. Mutsaers, S.E., Bishop, J.E., McGrouther, G., Laurent, G.J., 1997. Mechanisms of tissue repair: from wound healing to ®brosis. International Journal of Biochemical Cell Biology 29, 5±17. Platanias, L.C., Uddin, S., Domanski, P., Colamonici, O.R.,
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1996. Dierences in interferon a and b signaling. Journal of Biological Chemistry 271, 23630±23633. Ross, R.B., 1987. Treatment variables aecting facial growth in complete unilateral cleft lip and palate. Part 6. Techniques of palatal repair. Cleft Palate Journal 24, 64± 70. Sandhya, Rani M.R., Foster, G.R., Leung, S., Leaman, D., Stark, G.R., Ransoho, R.M., 1996. Characterization of beta-R1, a gene that is selectively induced by interferon beta compared with IFN-alpha. Journal of Biological Chemistry 271, 22878±22884. Searls, J.C., Kremenak, C.R., Rittman, B.R., 1979. Quantitative characterization of changes in cellularity and collagen ®ber size in contracting palatal wounds. Cleft Palate Journal 16, 373±380. Smith, T.J., Higgins, P.J., 1993. Interferon gamma regulation of de novo protein synthesis in human dermal ®broblasts in culture is anatomic site dependent. Journal of Investigative Dermatology 100, 288±292. Stephens, P., Davies, K.J., al-Khateeb, T., Shepherd, J.P., Thomas, D.W., 1996. A comparison of the ability of intraoral and extra-oral ®broblasts to stimulate extracellular matrix reorganization in a model of wound contraction. Journal of Dental Research 75, 1358±1364. Tredget, E.E., Shen, Y.J., Liu, G., Forsyth, N., Smith, C., Harrop, A.R., Scott, P.G., Ghahary, A., 1993. Regulation of collagen synthesis and messenger RNA levels in normal and hypertrophic scar ®broblasts in vitro by interferon alfa-2b. Wound Repair and Regeneration 1, 156±165. Witte, M.B., Barbul, A., 1997. General principles of wound healing. Surgical Clinics of North America 77, 509±528. Whitby, D.J., Ferguson, M.W.J., 1991a. The extracellular matrix of lip wounds in fetal, neonatal and adult mice. Development 12, 651±668. Whitby, D.J., Ferguson, M.W.J., 1991b. Immunohistological localisation of growth factors in fetal wound healing. Developmental Biology 147, 207±215.
Eects of interferons on proliferation and collagen synthesis of rat palatal wound ®broblasts A.M.H. Cornelissen*, J.W. Von den Ho, J.C. Maltha, A.M. KuijpersJagtman Department of Orthodontics and Oral Biology, College of Dental Sciences, Medical Faculty, University of Nijmegen, P.O. Box 9101, NL-6500 HB, Nijmegen, The Netherlands Accepted 23 February 1999
Abstract The purpose was to select drugs that speci®cally reduce collagen synthesis by palatal granulation ®broblasts without aecting their proliferation. Granulation ®broblasts were obtained from 8-day-old palatal mucoperiosteal wounds and normal ®broblasts from palatal tissue of unwounded rats. Cultured cells were treated with interferona2b, interferon-b and interferon-g (0, 100, 1000, and 10000 U/ml). Cell proliferation was measured by [3H]thymidine incorporation. Collagen synthesis and non-collagenous protein synthesis were determined from the incorporation of [3H]proline. None of the interferons signi®cantly inhibited the proliferation of either type of ®broblasts. Interferona2b had no eect on the variables studied at the dosages used. Interferon-b reduced collagen synthesis of granulation ®broblasts without aecting their non-collagenous protein synthesis or protein synthesis by normal ®broblasts. Interferon-g reduced collagen synthesis of both types of ®broblast and the non-collagenous protein synthesis of granulation ®broblasts. These data show that interferon-b speci®cally reduces collagen synthesis by oral granulation ®broblasts without aecting normal palatal ®broblasts. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Interferon; Wound healing; Oral ®broblasts; Collagen synthesis
1. Introduction Scar formation after cleft palate surgery is one of the major factors causing aberrant maxillary growth and dento-alveolar development (Ross, 1987). Consequently, the inhibition of scar formation seems a feasible way of improving the clinical outcome. Tissue injury triggers a complex cascade of cellular and biochemical events that ®nally results in a scar.
Abbreviations: DMEM, Dulbecco's modi®ed Eagle's medium; FCS, fetal calf serum; IFN, interferon; NEM, N-ethylmaleimide; TCA, trichloroacetic acid. * Corresponding author. Fax: +00-31-243-540631.
After injury, there is in¯ammation, ®broblast proliferation, matrix synthesis and remodelling (Witte and Barbul, 1997). Every phase contributes to the amount of scarring by in¯uencing the rate, quality and total amount of matrix deposition. Oral palatal wounds also pass through these phases of wound healing (Kahnberg and Thilander, 1982). The number of collagen ®bres in palatal mucoperiosteal wounds is increased up to 4 weeks after wounding (Searls et al., 1979). This seems to be due not only to an increased number of cells but also to an increase in collagen production per cell (Moriyama et al., 1991). A comparison of adult and foetal wound healing has shown that foetal wounds heal without scarring (Longaker and Adzick, 1991). This has been attributed
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to a lower in¯ammatory response (Whitby and Ferguson, 1991a) and a dierent cytokine pro®le (Whitby and Ferguson, 1991b), which suggests that manipulation of wound healing by altering the cytokine pro®le could lead to scarless healing. Only a few cytokines have been identi®ed that have the potential to reduce scarring; interferons represent the ®rst wellde®ned group of polypeptides that exert this property (Granstein et al., 1990a). The ability of interferons to modulate wound healing has been studied in vivo and on a variety of cell types in vitro. IFN-a, IFN-b and IFN-g reduce collagen synthesis by dermal ®broblasts (Duncan et al., 1995), hypertrophic scar ®broblasts (Tredget et al., 1993) and ®broblasts derived from sclerodermal lesions (Duncan and Berman, 1987). In vivo studies in mice have demonstrated that IFN-g reduced the increased collagen synthesis associated with the ®brotic response to an implanted foreign body (Granstein et al., 1990a). This eect was also found in bleomycin-induced pulmonary ®brosis and the healing response to cutaneous burn wounds (Granstein et al., 1990a). The therapeutic relevance of these in vitro and in vivo observations has been con®rmed in clinical trials. Intralesional injection of IFN-a2b (Berman and Duncan, 1989) or IFN-g (Granstein et al., 1990b) reduced the sizes of keloid and hypertrophic scars (Larrabee et al., 1990). The regulation of protein and collagen synthesis in normal ®broblasts by interferon in vitro depends on the anatomical site (Smith and Higgins, 1993; Martens et al., 1992). Moreover, dermal ®broblasts show a modulated response to IFN-g according to their stage of dierentiation during wound healing (Moulin et al., 1998). For wound healing factors such as migration (Irwin et al., 1994) and matrix remodelling (Stephens et al., 1996), it has been shown that there are phenotypic dierences between dermal and oral ®broblasts. Therefore it is quite possible that there are dierences in the regulation of proliferation and protein synthesis by interferons between dermal and oral ®broblasts, and between ®broblasts from palatal granulation tissue and normal palatal tissue. Our aim now was to determine the eects of interferons on proliferation, collagen synthesis, and noncollagenous protein synthesis of mucoperiosteal granulation and normal ®broblasts. 2. Materials and methods 2.1. Interferons Human recombinant IFN-a2b was obtained from Schering Plough BV (Amstelveen, The Netherlands). Fibroblast-derived human IFN-b was obtained from ICN Pharmaceuticals (Costa Mesa, USA) and rat
recombinant IFN-g was kindly provided by Dr P.H. van der Meide, (BPRC Rijswijk, The Netherlands). 2.2. Fibroblast cultures Five-week-old male Wistar rats were anaesthetized with a subcutaneous injection of 0.4 ml/kg body wt fentanyl+¯uanisone (Hypnorm1; Janssen-Cilaq, Beerse, Belgium). A standardized wound was made in the mucoperiosteum of the hard palate between the second molars using a 3-mm biopsy punch. The animals were medicated postoperatively with 0.1 mg/kg body wt buprenor®ne (Temgesic1; Schering Plough BV) as an analgesic. After 8 days, the rats were decapitated. Punch biopsy samples of the granulation tissue were collected in DMEM supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, 0.25 mg/ml fungizone, and 10% FCS, termed culture medium. Normal ®broblasts were obtained from biopsy samples of palatal mucosa of age-matched control rats. The samples of granulation tissue and normal mucoperiosteum were washed twice with culture medium and minced into small pieces. Primary ®broblast cultures were established from these explants. The cultures were maintained at 378C, 5% CO2 in culture medium in a humidi®ed incubator. Fibroblasts were passaged at con¯uence by trypsinization. All experiments were done on cells at low passage (<5). All culture supplies were purchased from Life Technologies (Breda, The Netherlands). 2.3. Proliferation assay Trypsinized ®broblasts were plated in 96-well microtitre plates at a density of 2000 cells/well in 100 ml culture medium. After 24 h, the cells were synchronized with culture medium containing 0.5% FCS for 24 h. Subsequently, the medium was replaced by 100 ml culture medium for 24 h. Then the medium was removed and replaced with 100 ml culture medium containing 420 kBq/ml [methyl-3H]thymidine (Amersham, Little Chalfont, UK) and various concentrations of IFNa2b, IFN-b, or IFN-g (0, 100, 1000, and 10000 U/ml). After 19 h, the medium was removed and the cells were washed three times with DMEM. Finally, the cells were harvested on a glass-®bre ®lter (LKBWallac, Turku, Finland) using a cell harvester (LKBWallac) and the incorporation of [methyl-3H]thymidine into DNA was counted with a betaplate liquid scintillation counter (LKB-Wallac). 2.4. Collagen and non-collagenous protein synthesis assay Collagen production by con¯uent monolayers was determined by the incorporation of [5-3H]proline into
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Table 1 Collagen and non-collagenous protein synthesis by palatal mucoperiosteal granulation tissue ®broblasts (GF) and normal palatal ®broblasts (NF)a
Collagen synthesis Non-collagenous protein synthesis a
counts/min per ng DNA counts/min per ng DNA
GF
NF
p
481.7250 821.3265
338.8244 660.8271
0.04 0.11
Data represent the mean value2SE of nine experiments.
bacterial collagenase-sensitive protein. The method described previously by Martens et al. (1992) was modi®ed to measure collagen synthesis in small numbers of ®broblasts. Trypsinized ®broblasts were dispensed into 24-well plates at a density of 20,000 cells/ well in 400 ml of medium and were grown until con¯uence. The medium was replaced with 400 ml of medium containing 50 mg/ml L-ascorbic acid (Sigma, St. Louis, USA), 164 kBq/ml L-[3H]proline (Amersham), and various concentrations of IFN-a2b, IFN-b, or IFN-g (0, 100, 1000, and 10000 U/ml). After the labelling period, cells and medium were collected and washed as described by Martens et al. (1992). The precipitate of cells and medium was dissolved in 550 ml 0.2 M NaOH at room temperature and neutralized with 445 ml 0.15 M HCl and 525 ml 1 M HEPES, pH 7.2 (Life Technologies). The solution was divided into three portions. (1) To determine the incorporation into total proteins, 200 ml was placed in a scintillation vial. (2) To determine the incorporation into collagen, 400 ml was placed in a 2-ml vial and digested by collagenase. (3) Another 400 ml was also placed in a 2-ml vial as a blank for the second portion. Collagenase digestion was performed by adding 400 ml highly puri®ed collagenase (ICN; 15 BTC units/ml) dissolved in 100 mM CaCl2H2O, 0.1 M TRIS and 2 mM NEM, pH 7.2. The blank digestion was done without collagenase. The second and third portions were incubated for 3 h at 378C. They were treated with 400 ml 15% TCA and 0.1% tannic acid (Sigma), centrifuged at 5000 g for 7 min, and the supernatant was placed in a scintillation vial. The pellet was resuspended and washed with 500 ml 5% TCA and 0.1% tannic acid. Aqua Luma (Lumac-LSC, Groningen, The Netherlands) was added as a scintillator, and the samples were measured by liquid scintillation counting (LKB-Wallac). Unless stated otherwise, the chemicals were obtained from Merck (Darmstad, Germany). The speci®city of this method was tested by labelling with [3H]tryptophan instead of [3H]proline, as trytophan is not present in collagen. The purity of the collagen fraction was 99.3%. The collagen synthesis was determined by subtraction of the counts released into the blank incubation
from those released in the presence of collagenase. The non-collagenous protein synthesis was calculated by subtraction of the radioactivity of the collagen synthesis from that of total protein. Collagen synthesis and non-collagenous protein synthesis were corrected for the amount of DNA per well with the PicoGreen1 dsDNA quantitation kit (Molecular Probes, Eugene, USA). 2.5. Statistical analysis Eighteen rats were used in total. From explants of nine wounded and nine control rats, nine separate cultures of granulation ®broblasts and nine separate cultures of normal ®broblasts were established. Three cultures of each type (granulation, normal) were used for every type of interferon (IFN-a2b, -b, -g). Each type of interferon was tested in triplicate cultures at a concentration of 0, 100, 1000, and 10000 U/ml. Values are expressed as the mean value relative to untreated cultures2SE. The collagen and non-collagenous protein synthesis of granulation ®broblasts was compared with that of normal ®broblasts by the student's t-test. The eects of the dierent concentrations of interferon (100, 1000 and 10000 U/ml) were compared with the control values (0 U/ml) by two-way analysis of variance on repeated measures. This was done in order to consider variations between ®broblasts from one rat to another. Signi®cant dierences were further analysed by Tukey's multiple-comparisons test. A p-value of less than 0.05 was considered to be statistically signi®cant. 3. Results 3.1. Collagen and non-collagenous protein synthesis of untreated ®broblasts Palatal granulation ®broblasts produced signi®cantly 42% more collagen than normal ®broblasts. Although not signi®cant, granulation ®broblasts produced 24% more non-collagenous proteins than normal ®broblasts (Table 1).
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Fig. 1. The eects of IFN-a2b on proliferation (A), collagen synthesis (B), and non-collagenous protein synthesis (C) of ®broblasts. Black bars represent palatal granulation-tissue ®broblasts and white bars normal ®broblasts. The results are expressed as percentage of untreated control cultures. Values are means2SE of three experiments. =p < 0.05.
Fig. 2. The eects of IFN-b on proliferation (A), collagen synthesis (B), and non-collagenous protein synthesis (C) of ®broblast. Black bars represent palatal granulation-tissue ®broblasts and white bars normal ®broblasts. The results are expressed as percentage of untreated control cultures. Values are means2SE of three experiments. =p < 0.05.
3.2. Interferon-a2b
collagenous protein synthesis in granulation ®broblasts decreased only by 624% and 1525% in the presence of 1000 and 10000 U/ml, respectively (Fig. 2C), which was not statistically signi®cant.
Although IFN-a2b tended to reduce the proliferation (Fig. 1A) of granulation and normal ®broblasts by about 10±20%, there were no signi®cant eects. Also, the collagen (Fig. 1B) and non-collagenous protein synthesis (Fig. 1C) of granulation and normal ®broblasts were not signi®cantly aected after incubation with IFN-a2b for 24 h. 3.3. Interferon-b IFN-b had no signi®cant eect on the proliferation of granulation or normal ®broblasts (Fig. 2A). Addition of IFN-b to cultures of normal ®broblasts did not aect collagen synthesis. By contrast, collagen synthesis by granulation ®broblasts was signi®cantly decreased, by 37213% and 3429% in the presence of 1000 and 10000 U/ml, respectively (Fig. 2B). The non-
3.4. Interferon-g IFN-g did not signi®cantly reduce the proliferation of granulation and normal ®broblasts (Fig. 3A). Collagen synthesis by granulation ®broblasts was signi®cantly reduced by 4727, 4326 and 4227% in the presence of 100, 1000, and 10000 U/ml, respectively (Fig. 3B). The suppression of collagen synthesis was not cell-type speci®c as synthesis by normal ®broblasts was also reduced by 31 2 4, 36 2 4, 31 2 8%, respectively. The eects of IFN-g on non-collagenous protein synthesis were more pronounced in granulation than in normal ®broblasts (Fig. 3C). A non-signi®cant suppression was found in normal ®broblasts, whereas a
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Fig. 3. The eects of IFN-g on proliferation (A), collagen synthesis (B), and non-collagenous protein synthesis (C) of ®broblasts. Black bars represent palatal granulation-tissue ®broblasts and white bars normal ®broblasts. The results are expressed as percentage of untreated control cultures. Values are means2SE of three experiments. =p < 0.05.
signi®cant decrease occurred in granulation ®broblasts by 3523, 3224 and 2727% in the presence of 100, 1000 and 10000 U/ml, respectively. 4. Discussion We have determined the eects of interferons on proliferation, collagen and non-collagenous protein synthesis of granulation-tissue ®broblasts. We have evidence that in this rat model the vast majority of ®broblasts in the granulation tissue are myo®broblasts at 8 days after wounding (A.M.H. Cornelissen et al., unpublished data). Myo®broblasts are involved in wound contraction, matrix synthesis, and remodelling. They have been associated with scar tissue formation (Estes et al., 1994), hypertrophic scarring and ®bromatosis (DesmoulieÁre and Gabbiani, 1996). Therefore we used cells derived from 8-day-old palatal mucoperios-
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teal wounds. The cells were not frozen and the number of passages was never more than ®ve, in order to preserve their original biosynthetic properties as much as possible. Fibroblast metabolism is modi®ed after wounding by other cells, the extracelllular matrix, and cytokines leading, for example, to an increased matrix synthesis (Mutsaers et al., 1997). We have found that rat palatal granulation ®broblasts cultured from 8-day-old wounds synthesize 42% more collagen than normal ®broblasts. In the same culture model, others have found that immature scar ®broblasts of 1-month-old palatal wounds produce 60% more collagen than normal palatal rat ®broblasts (Moriyama et al., 1991). Apparently, collagen production by mucoperiostealwound ®broblasts is upregulated for longer. It has recently been reported that human granulation ®broblasts taken from 12-day-old dermal wounds synthesize ®ve times more collagen than normal ®broblasts (Moulin et al., 1998). These ®ndings and our present results suggest that collagen synthesis by dermal ®broblasts after wounding is more upregulated than that of palatal ®broblasts. IFN-a, -b, and -g decrease the proliferation and collagen synthesis of ®broblasts derived from both normal and ®brotic skin (Duncan and Berman, 1985; Berman and Duncan, 1989; Tredget et al., 1993). The aim of our study was to compare the short-term eects of these interferons on proliferation, collagen and noncollagenous protein synthesis of palatal mucoperiosteal granulation or normal ®broblasts. We show that short-term exposure to IFN-a2b did not signi®cantly reduce the proliferation of palatal ®broblasts. A similar eect has been found in normal dermal ®broblasts (Duncan and Berman, 1985). Also, collagen synthesis by mucoperiosteal ®broblasts was not aected by IFN-a2b, which is in contrast with the suppressive eects of IFN-a2b on collagen synthesis by dermal ®broblasts (Duncan and Berman, 1985). This discrepancy may arise from dierences in type of ®broblasts but also from dierences in culture conditions. Although it is known that IFN-a stimulates immediate-response genes (Darnell et al., 1994), there are strong indications that it suppresses collagen synthesis by an indirect mechanism that takes approx. 48 h to have a signi®cant eect (Tredget et al., 1993). Therefore, it is possible that long-term exposure to IFN-a2b can reduce collagen synthesis by palatal ®broblasts. IFN-b had no eect on the proliferation of granulation or normal ®broblasts. It reduced collagen synthesis by granulation ®broblasts by about 35% and did not aect normal ®broblasts. It can be concluded that granulation ®broblasts dier from normal ®broblasts in their response to IFN-b. On the other hand, non-collagenous protein synthesis was not reduced by
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IFN-b in granulation and normal ®broblasts. These dierences in response to IFN-b between palatal granulation and normal ®broblasts are probably due to an intrinsic dierence as both cell types were treated in the same way. A dierential response of palatal scar ®broblasts and normal ®broblasts to cytokines such as transforming growth factor-b1, epidermal growth factor and platelet-derived growth factor has already been reported (Moriyama et al., 1991). IFN-g is generally known as an inhibitor of cell growth. We have found that it does not signi®cantly suppress the proliferation of palatal granulation or normal ®broblasts. Others reported that IFN-g stimulates the growth of human synovial ®broblasts and dental pulp ®broblasts (Butler et al., 1988; Melin et al, 1989). This discrepancy may arise from dierences in the type of ®broblasts but also from dierences in experimental conditions, such as the duration of treatment. Numerous studies have shown that IFN-g inhibits collagen synthesis by dermal ®broblasts, ®broblasts taken from ®brotic lesions (Granstein et al., 1990a) and dermal granulation ®broblasts (Moulin et al., 1998). In this respect, palatal normal and granulation ®broblasts behave the same. IFN-g regulates collagen synthesis by reducing the transcription rate of the COL1A1 and COL3A1 gene, the stability of the corresponding transcript and the rate of translation (Diaz and Jimenez, 1997). In contrast to IFN-b, IFN-g suppressed collagen synthesis by palatal granulation ®broblasts non-speci®cally, as non-collagenous protein synthesis was also reduced. Others have found that IFN-g does not aect non-collagenous protein synthesis of dermal granulation ®broblasts (Moulin et al., 1998). This discrepancy indicates that palatal granulation ®broblasts are perhaps more sensitive to IFN-g than dermal granulation ®broblasts. However, the dierence could also be related to dierences in culture conditions. Variations in potency to inhibit collagen synthesis between the interferons might be explained by the mechanisms of signal transduction. IFN-g (type II) receptors on ®broblasts have been demonstrated to be dierent from the type I interferon receptors. Although IFN-a and IFN-b both bind to the type I receptor (Darnell et al., 1994), they transduce dierent signals (Platanias et al., 1996) and can induce dierent genes (Sandhya Rani et al., 1996). In conclusion, IFN-b and IFN-g are both able to reduce collagen synthesis by palatal granulation ®broblasts in vitro. An optimal therapy against palatal scarring reduces the collagen synthesis of ®broblasts without aecting the synthesis of other proteins and without aecting normal ®broblasts. This means that IFN-b is the more appropriate interferon for further studies on scar reduction in vivo.
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