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Effects of β-glucan on proliferation and migration of fibroblasts
Current Applied Physics 5 (2005) 468–471 www.elsevier.com/locate/cap
Effects of b-glucan on proliferation and migration of fibroblasts Hyun Joo Son a
a,b
, Hyun Chul Bae a,b, Hyun Jeong Kim a, Dong Hee Lee a, Dong-Wook Han a, Jong-Chul Park a,b,*
Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea b Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea Received 18 October 2004; received in revised form 10 November 2004 Available online 25 February 2005
Abstract b-Glucans, originated from the outer cell wall of fungi, are shown to have immune stimulatory activity, especially to enhance wound healing. In the wound healing processes, the migration and proliferation of fibroblasts are essential. Actually fetal and neonatal wounds heal without scar because the migration of those dermal fibroblasts is faster than that of adult fibroblasts. Furthermore, it has been reported that human dermal fibroblasts express receptors for b-glucans like macrophages. In this study, the direct effects of b-glucans on the proliferation and migration of L-929 fibroblasts were examined in vitro. b-Glucan treatment to the L-929 cells with 0.5 or 5 mg/ml resulted in a significant (p < 0.05) dose-dependant increase in cell proliferation. However, the migration assay using a self-designed cell tracking system showed that there was no significant difference in the migration speed between the glucan-treated and non-treated cells. These results suggest that b-glucans can enhance directly fibroblast proliferation in vitro, but not affect fibroblast migration. 2005 Elsevier B.V. All rights reserved. PACS: 87.14.g; 87.17.Jj; 87.80.Rb Keywords: b-Glucan; Wound healing; Fibroblast; Migration; Proliferation
1. Introduction Wound repair is a complex physiological process that requires the interaction of cells with diverse phenotypes, such as macrophages, fibroblasts, etc., and is mainly mediated by immune reaction [1]. In the wound healing mechanisms, there have been two major hypotheses about fibroblast migration and proliferation. One is that some signals created after the occurrence of wound activate macrophage to release cytokines much more, and these cytokines signal fibroblasts to migrate into wound sites and to proliferate [2,3]. Another hypothesis about wound healing is that some chemicals from wound di*
Corresponding author. Fax: +82 2 363 9923. E-mail address: [email protected] (J.-C. Park).
1567-1739/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cap.2005.01.011
rectly signal fibroblasts to migrate and proliferate, through cellular receptor-mediated signaling. Clinically it has been well-known that fetal and neonatal wounds heal without contraction and scar formation as the fetal and neonatal dermal fibroblasts migrate faster into wound sites than adult dermal fibroblasts [4]. It has been reported that (1 ! 6) branched (1 ! 3)-bD-glucans, polymers of glucose, mainly construct the outer cell wall of fungi and have wound healing activity [5–7]. The biological activity of b-glucan is mediated through pattern recognition receptors located on target cells. Moreover, recent studies showed the existence of b-glucan binding sites on normal human dermal fibroblast other than immunocytes [8,9]. In terms of the immune stimulatory activity of b-glucan related with wound healing, there are two possible action modes;
H.J. Son et al. / Current Applied Physics 5 (2005) 468–471
one is indirect activation through various cytokines of macrophages and the other is direct influence on fibroblasts. Since the former would be mostly in vivo situation, the present study was focused on the direct effects of (1 ! 3), (1 ! 6)-b-D-glucans on the proliferation and migration of fibroblasts in vitro.
469
instruction, the cells were incubated with WST-8 in the last 4 h of the culture period tested at 37 C in the dark. The absorbance was determined at 450 nm in an ELISA reader (Spectra Max 340, Molecular Device Co., Sunnyvale, CA, USA). 2.3. Cell migration assay
2. Materials and methods 2.1. b-Glucan and cell culture (1 ! 3)-b-D-Glucans (source: Letinulus edodes) that have b-D-glucopyranosyl units attached by (1 ! 6) linkages as single unit branches were obtained from WAKO Pure Chemical Industry (Osaka, Japan). L929 cells (from mouse connective tissue) were obtained from American Type Culture Collection (Rockville, MD, USA). The cells were routinely maintained in DulbeccoÕs modified EagleÕs medium (Sigma Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma Co.) and 1% antibiotic antimycotic solution (Sigma Co.) at 37 C and 5% CO2 in a humid environment. 2.2. Cell proliferation assay L-929 cells with 1 · 105 cells/ml of initial seeding density were grown at 80% confluence and further cultured under serum free conditions for 24 h. Afterwards, b-glucans were treated to the cells at a final concentration of 0.5 or 5.0 mg/ml and cultured for 3 days. The number of viable cells was measured indirectly using a highly water soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Dojindo Lab., Kumamoto, Japan), reduced to formazan dye by mitochondrial dehydrogenase [10]. According to manufacturerÕs
The migration assay using a self-designed cell tracking system was performed as previously described [4]. As shown in Fig. 1, L-929 cells (2 · 103 cells/ml) were seeded in the hole (400 lm in diameter) of silicon rubber attached to the bottom of chambered cover-glass slide. After 1 h of incubation, the silicon rubber was removed and 1 mg/ml b-glucan was treated to the attached cells. The cells were observed in the self-designed CO2 miniincubator placed on the microscope stage for 24 h, and the images were captured from the CCD camera attached to the microscope. The edge point of each cell was detected, and the migration of the center was recorded with cell movement path, accumulated distance and speed. 2.4. Statistical analysis All the variables were tested in two independent cultures for each experiment, and each experiment was repeated duplicate (n = 4). The results were reported as a mean ± standard deviation and analyzed by Student ttests. Statistical significance was considered at p < 0.05.
3. Results and discussion It was shown that the proliferation of L-929 cells treated with 5.0 mg/ml b-glucan was significantly (p < 0.05) increased compared to that of the non-treated control (Fig. 2). This result suggests that ligation of
Fig. 1. Schematic illustration for procedure of cell migration assay using self-designed cell tracking system.
470
H.J. Son et al. / Current Applied Physics 5 (2005) 468–471
Fig. 2. Effects of b-glucan on proliferation of L-929 cells. The results are reported as a mean ± standard deviation (n = 4). The data is analyzed by Student t-tests, and the values marked with asterisks are significantly (p < 0.05) different from the non-treated control.
b-glucan by its cognate receptors on fibroblasts enhances the cell proliferation. Moreover, it has been reported that there were specific receptors for glucans on human fibroblasts, especially found in the wound milieu [9]. The results of the present study could be also confirmed by the evidence showing that a highly purified, well characterized glucan stimulated the procollagen gene expression and collagen biosynthesis of fibroblasts [11]. During wound healing, fibroblasts synthesize and later remodel new extracellular matrix material, of which the main component are collagen I and III, which increase the tensile strength of the wound [2,12,13]. These results are supported by clinical report showing that pediatric burns could be effectively treated with glucan–collagen mixtures and this preparation markedly simplified wound care and significantly decreased postinjury pain. These observations suggest that glucans and related immunomodulators may be useful adjuncts for healing, particularly in burn wounds. The cell migration assay showed that when the cells treated with 1.0 mg/ml b-glucan, the accumulated distance was 1180 lm and almost similar with that of the non-treated control (1220 lm, Fig. 3). Furthermore, it was demonstrated that the average migration speed of the glucan-treated cells was 48.90 lm/h, while that of the non-treated control was 52.65 lm/h (Fig. 4). There were no significant differences in the accumulated distance and the migration speed between the glucan-treated and non-treated cells. These results suggest that the present migration assay using the in vitro cell tracking system can not determine whether b-glucan directly affects the cell migration or not. It is well known that the macrophage is a pivotal cell in wound repair and there are pattern recognition receptors for glucans on mammalian macrophages. Nevertheless, the mechanism by which b-glucan or other immunomodulators enhance wound repair has remained elusive. From the present results, it might be speculated that a direct interaction of
Fig. 3. Effects of b-glucan on accumulated movement distances of L929 cells.
Fig. 4. Effects of b-glucan on average migration speed of L-929 cells. The results are reported as a mean ± standard deviation (n = 4). The data is analyzed by Student t-tests, and there is no significant difference between the glucan-treated and non-treated cells.
the b-glucan with fibroblasts was proposed as a possible mechanism of wound repair while the effect of b-glucan on repair involved macrophage release of wound growth factors with subsequent modulation of fibroblast activity including collagen biosynthesis.
Acknowledgment This study was supported by the Ministry of Science and Technology of the Republic of Korea (Grant no. R01-2004-000-10580-0).
References [1] L.A. DiPietro, Shock 4 (1995) 233. [2] A.J. Singer, R.A. Clark, New Engl. J. Med. 341 (1999) 738.
H.J. Son et al. / Current Applied Physics 5 (2005) 468–471 [3] W.T. Lawrence, R.F. Diegelmann, Clin. Dermatol. 12 (1994) 157. [4] J.-C. Park, B.J. Park, H. Suh, B.Y. Park, D.K. Rah, Yonsei Med. J. 42 (2001) 587. [5] B.A. Stone, A.E. Clarke, Chemistry and Biology of (1–3)bglucans, LaTrobe University Press, Melbourne, 1992. [6] D.L. Williams, Mediat. Inflamm. 6 (1997) 247. [7] S.J. Delatte, J. Evans, A. Hebra, W. Adamson, H.B. Othersen, E.P. Tagge, J. Pediatr. Surg. 36 (2001) 113. [8] A. Mueller, J. Raptis, P.J. Rice, J.H. Kalbfleisch, R.D. Stout, H.E. Ensley, W. Browder, D.L. Williams, Glycobiology 10 (2000) 339.
471
[9] P. Kougias, D. Wei, P.J. Rice, H.E. Ensley, J. Kalbfleisch, D.L. Williams, I.W. Browder, Infect. Immun. 69 (2001) 3933. [10] Y. Kanemura, H. Mori, S. Kobayashi, O. Islam, E. Kodama, A. Yamamoto, Y. Nakanishi, N. Arita, M. Yamasaki, H. Okano, M. Hara, J. Miyake, J. Neurosci. Res. 69 (2002) 869. [11] D. Wei, L. Zhang, L. David, D.L. Williams, I.W. Browder, Wound Repair Regen. 10 (2002) 161. [12] S.E. Mutsaers, J.E. Bishop, G. McGroyther, G.J. Laurent, Int. J. Biochem. Cell. Biol. 29 (1997) 5. [13] M. Eastwood, R. Porter, U. Khan, G. McGroythe, R. Brown, J. Cell. Physiol. 166 (1996) 33.
Effects of b-glucan on proliferation and migration of fibroblasts Hyun Joo Son a
a,b
, Hyun Chul Bae a,b, Hyun Jeong Kim a, Dong Hee Lee a, Dong-Wook Han a, Jong-Chul Park a,b,*
Department of Medical Engineering, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea b Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea Received 18 October 2004; received in revised form 10 November 2004 Available online 25 February 2005
Abstract b-Glucans, originated from the outer cell wall of fungi, are shown to have immune stimulatory activity, especially to enhance wound healing. In the wound healing processes, the migration and proliferation of fibroblasts are essential. Actually fetal and neonatal wounds heal without scar because the migration of those dermal fibroblasts is faster than that of adult fibroblasts. Furthermore, it has been reported that human dermal fibroblasts express receptors for b-glucans like macrophages. In this study, the direct effects of b-glucans on the proliferation and migration of L-929 fibroblasts were examined in vitro. b-Glucan treatment to the L-929 cells with 0.5 or 5 mg/ml resulted in a significant (p < 0.05) dose-dependant increase in cell proliferation. However, the migration assay using a self-designed cell tracking system showed that there was no significant difference in the migration speed between the glucan-treated and non-treated cells. These results suggest that b-glucans can enhance directly fibroblast proliferation in vitro, but not affect fibroblast migration. 2005 Elsevier B.V. All rights reserved. PACS: 87.14.g; 87.17.Jj; 87.80.Rb Keywords: b-Glucan; Wound healing; Fibroblast; Migration; Proliferation
1. Introduction Wound repair is a complex physiological process that requires the interaction of cells with diverse phenotypes, such as macrophages, fibroblasts, etc., and is mainly mediated by immune reaction [1]. In the wound healing mechanisms, there have been two major hypotheses about fibroblast migration and proliferation. One is that some signals created after the occurrence of wound activate macrophage to release cytokines much more, and these cytokines signal fibroblasts to migrate into wound sites and to proliferate [2,3]. Another hypothesis about wound healing is that some chemicals from wound di*
Corresponding author. Fax: +82 2 363 9923. E-mail address: [email protected] (J.-C. Park).
1567-1739/$ - see front matter 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cap.2005.01.011
rectly signal fibroblasts to migrate and proliferate, through cellular receptor-mediated signaling. Clinically it has been well-known that fetal and neonatal wounds heal without contraction and scar formation as the fetal and neonatal dermal fibroblasts migrate faster into wound sites than adult dermal fibroblasts [4]. It has been reported that (1 ! 6) branched (1 ! 3)-bD-glucans, polymers of glucose, mainly construct the outer cell wall of fungi and have wound healing activity [5–7]. The biological activity of b-glucan is mediated through pattern recognition receptors located on target cells. Moreover, recent studies showed the existence of b-glucan binding sites on normal human dermal fibroblast other than immunocytes [8,9]. In terms of the immune stimulatory activity of b-glucan related with wound healing, there are two possible action modes;
H.J. Son et al. / Current Applied Physics 5 (2005) 468–471
one is indirect activation through various cytokines of macrophages and the other is direct influence on fibroblasts. Since the former would be mostly in vivo situation, the present study was focused on the direct effects of (1 ! 3), (1 ! 6)-b-D-glucans on the proliferation and migration of fibroblasts in vitro.
469
instruction, the cells were incubated with WST-8 in the last 4 h of the culture period tested at 37 C in the dark. The absorbance was determined at 450 nm in an ELISA reader (Spectra Max 340, Molecular Device Co., Sunnyvale, CA, USA). 2.3. Cell migration assay
2. Materials and methods 2.1. b-Glucan and cell culture (1 ! 3)-b-D-Glucans (source: Letinulus edodes) that have b-D-glucopyranosyl units attached by (1 ! 6) linkages as single unit branches were obtained from WAKO Pure Chemical Industry (Osaka, Japan). L929 cells (from mouse connective tissue) were obtained from American Type Culture Collection (Rockville, MD, USA). The cells were routinely maintained in DulbeccoÕs modified EagleÕs medium (Sigma Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma Co.) and 1% antibiotic antimycotic solution (Sigma Co.) at 37 C and 5% CO2 in a humid environment. 2.2. Cell proliferation assay L-929 cells with 1 · 105 cells/ml of initial seeding density were grown at 80% confluence and further cultured under serum free conditions for 24 h. Afterwards, b-glucans were treated to the cells at a final concentration of 0.5 or 5.0 mg/ml and cultured for 3 days. The number of viable cells was measured indirectly using a highly water soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] (Dojindo Lab., Kumamoto, Japan), reduced to formazan dye by mitochondrial dehydrogenase [10]. According to manufacturerÕs
The migration assay using a self-designed cell tracking system was performed as previously described [4]. As shown in Fig. 1, L-929 cells (2 · 103 cells/ml) were seeded in the hole (400 lm in diameter) of silicon rubber attached to the bottom of chambered cover-glass slide. After 1 h of incubation, the silicon rubber was removed and 1 mg/ml b-glucan was treated to the attached cells. The cells were observed in the self-designed CO2 miniincubator placed on the microscope stage for 24 h, and the images were captured from the CCD camera attached to the microscope. The edge point of each cell was detected, and the migration of the center was recorded with cell movement path, accumulated distance and speed. 2.4. Statistical analysis All the variables were tested in two independent cultures for each experiment, and each experiment was repeated duplicate (n = 4). The results were reported as a mean ± standard deviation and analyzed by Student ttests. Statistical significance was considered at p < 0.05.
3. Results and discussion It was shown that the proliferation of L-929 cells treated with 5.0 mg/ml b-glucan was significantly (p < 0.05) increased compared to that of the non-treated control (Fig. 2). This result suggests that ligation of
Fig. 1. Schematic illustration for procedure of cell migration assay using self-designed cell tracking system.
470
H.J. Son et al. / Current Applied Physics 5 (2005) 468–471
Fig. 2. Effects of b-glucan on proliferation of L-929 cells. The results are reported as a mean ± standard deviation (n = 4). The data is analyzed by Student t-tests, and the values marked with asterisks are significantly (p < 0.05) different from the non-treated control.
b-glucan by its cognate receptors on fibroblasts enhances the cell proliferation. Moreover, it has been reported that there were specific receptors for glucans on human fibroblasts, especially found in the wound milieu [9]. The results of the present study could be also confirmed by the evidence showing that a highly purified, well characterized glucan stimulated the procollagen gene expression and collagen biosynthesis of fibroblasts [11]. During wound healing, fibroblasts synthesize and later remodel new extracellular matrix material, of which the main component are collagen I and III, which increase the tensile strength of the wound [2,12,13]. These results are supported by clinical report showing that pediatric burns could be effectively treated with glucan–collagen mixtures and this preparation markedly simplified wound care and significantly decreased postinjury pain. These observations suggest that glucans and related immunomodulators may be useful adjuncts for healing, particularly in burn wounds. The cell migration assay showed that when the cells treated with 1.0 mg/ml b-glucan, the accumulated distance was 1180 lm and almost similar with that of the non-treated control (1220 lm, Fig. 3). Furthermore, it was demonstrated that the average migration speed of the glucan-treated cells was 48.90 lm/h, while that of the non-treated control was 52.65 lm/h (Fig. 4). There were no significant differences in the accumulated distance and the migration speed between the glucan-treated and non-treated cells. These results suggest that the present migration assay using the in vitro cell tracking system can not determine whether b-glucan directly affects the cell migration or not. It is well known that the macrophage is a pivotal cell in wound repair and there are pattern recognition receptors for glucans on mammalian macrophages. Nevertheless, the mechanism by which b-glucan or other immunomodulators enhance wound repair has remained elusive. From the present results, it might be speculated that a direct interaction of
Fig. 3. Effects of b-glucan on accumulated movement distances of L929 cells.
Fig. 4. Effects of b-glucan on average migration speed of L-929 cells. The results are reported as a mean ± standard deviation (n = 4). The data is analyzed by Student t-tests, and there is no significant difference between the glucan-treated and non-treated cells.
the b-glucan with fibroblasts was proposed as a possible mechanism of wound repair while the effect of b-glucan on repair involved macrophage release of wound growth factors with subsequent modulation of fibroblast activity including collagen biosynthesis.
Acknowledgment This study was supported by the Ministry of Science and Technology of the Republic of Korea (Grant no. R01-2004-000-10580-0).
References [1] L.A. DiPietro, Shock 4 (1995) 233. [2] A.J. Singer, R.A. Clark, New Engl. J. Med. 341 (1999) 738.
H.J. Son et al. / Current Applied Physics 5 (2005) 468–471 [3] W.T. Lawrence, R.F. Diegelmann, Clin. Dermatol. 12 (1994) 157. [4] J.-C. Park, B.J. Park, H. Suh, B.Y. Park, D.K. Rah, Yonsei Med. J. 42 (2001) 587. [5] B.A. Stone, A.E. Clarke, Chemistry and Biology of (1–3)bglucans, LaTrobe University Press, Melbourne, 1992. [6] D.L. Williams, Mediat. Inflamm. 6 (1997) 247. [7] S.J. Delatte, J. Evans, A. Hebra, W. Adamson, H.B. Othersen, E.P. Tagge, J. Pediatr. Surg. 36 (2001) 113. [8] A. Mueller, J. Raptis, P.J. Rice, J.H. Kalbfleisch, R.D. Stout, H.E. Ensley, W. Browder, D.L. Williams, Glycobiology 10 (2000) 339.
471
[9] P. Kougias, D. Wei, P.J. Rice, H.E. Ensley, J. Kalbfleisch, D.L. Williams, I.W. Browder, Infect. Immun. 69 (2001) 3933. [10] Y. Kanemura, H. Mori, S. Kobayashi, O. Islam, E. Kodama, A. Yamamoto, Y. Nakanishi, N. Arita, M. Yamasaki, H. Okano, M. Hara, J. Miyake, J. Neurosci. Res. 69 (2002) 869. [11] D. Wei, L. Zhang, L. David, D.L. Williams, I.W. Browder, Wound Repair Regen. 10 (2002) 161. [12] S.E. Mutsaers, J.E. Bishop, G. McGroyther, G.J. Laurent, Int. J. Biochem. Cell. Biol. 29 (1997) 5. [13] M. Eastwood, R. Porter, U. Khan, G. McGroythe, R. Brown, J. Cell. Physiol. 166 (1996) 33.