The Journal of Dermatology Vol. 19: 6!'i4-656, 1992
WS 1-5
Alteration of the Chemotactic Response of Human Skin Fibroblasts to PDGF by Growth Factors Yoshinao Soma, Kazuhiko Takehara and Yasumasa Ishibashi 100...-------------------,
Introduction In the process of wound healing, the migration of cells, including inflammatory cells, endothelial cells, and fibroblasts, is essential and critical. This migration is thought to be determined in large part by the production of chemoattractants. Collagen and collagen-like peptides (1), fibronectin (2), TGF-IJ (3), IL-4 (4) and PDGF (5) have been reported to be chemotactic for fibroblastic cells. PDGF was originally described as a mitogen for smooth muscle cells (6). In 1982, Seppa et al. (5) reported that PDGF is not only a mitogen but also a potent chemoattractant for fibroblasts. Other growth factors, including EGF and FGF, are not active as chemoattractants. In contrast to previous report (3), TGF-IJ was not chemotactic for human skin fibroblasts in our experiment, as shown in Figure 1. Thus, PDGF may be the only growth factor that is a chemoattractant for fibroblasts. An investigation into the conditions which alter the chemotactic response ofNIH/3T3 cells to PDGF was made by Grotendorst in 1984 (7). He reported that quiescent cells respond much better than exponentially growing cells and that preincubation of the cells with mitogens, including EGF, FGF, PDGF, and serum, for 3 hours inhibits the chemotactic response. Because NIH/3T3 cells are an immortal cell line, they may behave differently in their chemotactic response to PDGF from diploid cells of primary culture. Since there has been no report examining the specific alteration of the chemotactic response of primary culture of human skin fibroblasts, we have investigated factors that alter the migratory response of human skin fibroblasts to PDGF. Materials and Methods Cell Culture Human skin fibroblasts were grown from exDepartment of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
o
TGF-BETA
0---4
~
PDGF
0--0
...J
3 ...J
~
0.01
0.1
10
GROWTH FACTOR CONCENTRATION (ng/ml)
Fig. 1. Chemotactic response of human skin fibroblasts to PDGF and TGF,/3 was determined as described under Materials and Methods. PDGF was chemotactic in a dose dependent manner; TGF-/3 was not.
plants of healthy human adult skin and cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Cells were used for the experiments prior to the third passage. Growth Factors PDGF and TGF-beta were purchased from R&D Inc. EGF and basic FGF were purchased from Sigma Chemical Company. Chemotaxis Assay The chemotaxis assay was performed by using blind well chambers as described previously (8). The test substance was diluted in MEM containing 0.2 mg/ml BSA and added to the lower well of each blind well chamber. This lower well was than covered with a collagen-coated polycarbonate filter (Nucleopore, 8 micrometer-diameter pores). The upper well was then fixed in place, and cells freshly released from tissue culture flasks were added to it in MEM containing 2 mg/ml BSA. After 4 hours of incubation at 37°C in 90% air and 10% CO 2, the filters were removed and the cells were fixed and stained by Diff-Quick stain. The upper surface cells
Alteration of the Chemotactic Response to PDGF
655
were removed by scraping with a rubber policeman, and the number of cells on the lower surface of the filter was determined by counting the number of nuclei (Fig. 2). Results
Effea of Growth State on the Chemotactic Response Different numbers of human skin fibroblasts were placed in 5 culture flasks (flask I to flask 5) and incubated in MEM/I0% FBS for 6 days. Then the cells were trypsinized, cell numbers counted, and chemotactic responses to PDGF were tested. Cell densities in flasks I to 5 were 1.52, 2.56, 3.12, 4.28 and 5.00x 104/cm 2, respectively. The cells in flasks I to 3 were growing exponentially and those in flasks 4 and 5 were apparently confluent. The cells in flask 2 presented the maximal response to PDGF. The migratory responses of the cells in flasks 4 and 5 were lower than the others. These results suggested that exponentially growing cells responded better than confluent ones, in contrast to a previous report (7) that confluent cells responded better than exponentially growing ones.
Effed of Serum Treatment on the Chemotactic Response Next we wanted to determine the effect of serum treatment on the chemotactic response of human skin fibroblasts, since a previous paper (7) reported that 3 hours treatment with serum decreased the chemotactic response of NIH/3T3 cells to PDGF. Confluent cultures of human skin fibroblasts were growth arrested by incubation in serum-free MEM/2/mg/ml BSA for 3 days. Then the media were changed to MEM/I0% FBS. After 3, 8, 24, and 48 hours, their chemotactic responses to PDGF were determined. The chemotactic response to PDGF after exposure to FBS for 3 hours was markedly decreased (>50%), and the inhibitory effect of serum lasted for 24 hours. These results were similar to those of the previous report by Grotendorst (7).
Effect of Growth Factors on the Chemotadic Response Since serum is a mixture of many mitogens, we investigated whether any purified mitogens contained in serum could affect the chemotactic response of human skin fibroblasts to PDGF. Confluent cultures of human skin fibroblasts were incubated in serum-free MEM/BSA for 4 days. Then growth factors, including PDGF, FGF, EGF, and TGF-J3, were added separately to final concentrations of 20 ng/ml. After 3 and 24 hours, the . chemotactic responses to PDGF were tested. Three and 24 hours of preincubation with EGF showed inhibitory effects on the chemotactic response to
Fig. 2. Chemotaxis assay was performed and the filters were observed microscopically. 20 ng/ml of PDCF [PDGF (+)] or no PDGF [PDGF (-)] was added to the lower chamber.
PDGF. FGF also decreased the chemotactic response after 24 hours. The data presented here are consistent with the previous report that FGF and EGF inhibited the migratory response of NIH/3T3 cells to PDGF (7). Preincubation with TGF-J3 for 3 hours did not have any effect on the migratory response of human skin fibroblasts. However, 24 hours of preincubation with TGF-p markedly enhanced both the migration toward PDGF and the basal level of random migration in the control chamber without PDGF. The stimulatory effect of TGF-p on the cell migration may be attributed to enhanced cell attachment to the filter as a result of increased matrix synthesis induced by TGF-p. Discussion
In the process of wound healing, many kinds of biologically active substances are involved in a complex manner and considered to play key roles in the regulation of cells' proliferation, migration, and protein synthesis. Among those factors, PDGF is
656
Soma et al
one of the most important chemoattractants for connective tissue cells. The data presented here indicated that the migratory response to PDGF is regulated not only by the gradient of PDGF concentration, but also by other mitogens and the growth state of the cells. Grotendorst (7) reported that NIH/3T3 cells that are stimulated by mitogens show a decreased chemotactic response to PDGF and suggested that cells that are committed to entering the cell cycle lose their ability to migrate in response to PDGF. His observations were supported by our data presented here that human skin fibroblasts exposed to mitogens including EGF, FGF, and serum showed decreased chemotactic responses to PDGF. However, in sharp contrast to NIH/3T3 cells, human skin fibroblasts migrated to PDGF more when the cells were exponentially growing, suggesting that the chemotactic response of human skin fibroblasts may be regulated differently from that of fibroblasts from an immortal cell line. The observations that growing cells showed a high ability to migrate toward PDGF may account for the simultaneous stimulation of proliferation and migration of connective tissue cells in the early stages of wound healing.
References 1) Postlethwaite AE, Seyer LM, Kang AH: Chemotactic attraction of human fibroblasts to type I, II and III collagens and collagen-derived peptides, Proc Natl Acad Sci USA, 75: 871-875, 1978. 2) Gauss-Muller, Kleinman HK, Martin GR, Schiffman E: Role of attachment factors and attractants in fibroblast chemotaxis,] Lab Clin Med, 96: 1071-1080, 1980. 3) Postlethwaite AE, Keski-Oja], Moses HL, Kang AH: Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor-p,] &p Med, 165: 251-256, 1987. 4) Postlethwaite AE, Seyer LM: Fibroblast chemotaxis induction by human recombinant interleukin-4,] Clin Invest, 87: 2147-2152, 1991. 5) Seppa HE], Grotendorst GR, Seppa SI, Shiffman E, Martin GR: The platelet-derived growth factor is a chemoattractant for fibroblasts,] Cell Bioi, 92: 584588,1982. 6) Ross R, Raines EW, Bowen-Pope DF: The biology of platelet-derived growth factor, Cell, 46: 155-169,1986. 7) Grotendorst GR: Alteration of the chemotactic response ofNIH/3T3 cells to PDGFby growth factors, transformation, and tumor promoters, Cell, 36: 279285,1984. 8) Grotendorst GR, Seppa HE;J, Kleinman HK, Martin GR: Attachment of smooth muscle cells to collagen and their migration toward platelet-derived growth factor, Proc Natl &.ad Sci USA, 78: 3669-3672, 1981.
WS 1-5
Alteration of the Chemotactic Response of Human Skin Fibroblasts to PDGF by Growth Factors Yoshinao Soma, Kazuhiko Takehara and Yasumasa Ishibashi 100...-------------------,
Introduction In the process of wound healing, the migration of cells, including inflammatory cells, endothelial cells, and fibroblasts, is essential and critical. This migration is thought to be determined in large part by the production of chemoattractants. Collagen and collagen-like peptides (1), fibronectin (2), TGF-IJ (3), IL-4 (4) and PDGF (5) have been reported to be chemotactic for fibroblastic cells. PDGF was originally described as a mitogen for smooth muscle cells (6). In 1982, Seppa et al. (5) reported that PDGF is not only a mitogen but also a potent chemoattractant for fibroblasts. Other growth factors, including EGF and FGF, are not active as chemoattractants. In contrast to previous report (3), TGF-IJ was not chemotactic for human skin fibroblasts in our experiment, as shown in Figure 1. Thus, PDGF may be the only growth factor that is a chemoattractant for fibroblasts. An investigation into the conditions which alter the chemotactic response ofNIH/3T3 cells to PDGF was made by Grotendorst in 1984 (7). He reported that quiescent cells respond much better than exponentially growing cells and that preincubation of the cells with mitogens, including EGF, FGF, PDGF, and serum, for 3 hours inhibits the chemotactic response. Because NIH/3T3 cells are an immortal cell line, they may behave differently in their chemotactic response to PDGF from diploid cells of primary culture. Since there has been no report examining the specific alteration of the chemotactic response of primary culture of human skin fibroblasts, we have investigated factors that alter the migratory response of human skin fibroblasts to PDGF. Materials and Methods Cell Culture Human skin fibroblasts were grown from exDepartment of Dermatology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan.
o
TGF-BETA
0---4
~
PDGF
0--0
...J
3 ...J
~
0.01
0.1
10
GROWTH FACTOR CONCENTRATION (ng/ml)
Fig. 1. Chemotactic response of human skin fibroblasts to PDGF and TGF,/3 was determined as described under Materials and Methods. PDGF was chemotactic in a dose dependent manner; TGF-/3 was not.
plants of healthy human adult skin and cultured in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS). Cells were used for the experiments prior to the third passage. Growth Factors PDGF and TGF-beta were purchased from R&D Inc. EGF and basic FGF were purchased from Sigma Chemical Company. Chemotaxis Assay The chemotaxis assay was performed by using blind well chambers as described previously (8). The test substance was diluted in MEM containing 0.2 mg/ml BSA and added to the lower well of each blind well chamber. This lower well was than covered with a collagen-coated polycarbonate filter (Nucleopore, 8 micrometer-diameter pores). The upper well was then fixed in place, and cells freshly released from tissue culture flasks were added to it in MEM containing 2 mg/ml BSA. After 4 hours of incubation at 37°C in 90% air and 10% CO 2, the filters were removed and the cells were fixed and stained by Diff-Quick stain. The upper surface cells
Alteration of the Chemotactic Response to PDGF
655
were removed by scraping with a rubber policeman, and the number of cells on the lower surface of the filter was determined by counting the number of nuclei (Fig. 2). Results
Effea of Growth State on the Chemotactic Response Different numbers of human skin fibroblasts were placed in 5 culture flasks (flask I to flask 5) and incubated in MEM/I0% FBS for 6 days. Then the cells were trypsinized, cell numbers counted, and chemotactic responses to PDGF were tested. Cell densities in flasks I to 5 were 1.52, 2.56, 3.12, 4.28 and 5.00x 104/cm 2, respectively. The cells in flasks I to 3 were growing exponentially and those in flasks 4 and 5 were apparently confluent. The cells in flask 2 presented the maximal response to PDGF. The migratory responses of the cells in flasks 4 and 5 were lower than the others. These results suggested that exponentially growing cells responded better than confluent ones, in contrast to a previous report (7) that confluent cells responded better than exponentially growing ones.
Effed of Serum Treatment on the Chemotactic Response Next we wanted to determine the effect of serum treatment on the chemotactic response of human skin fibroblasts, since a previous paper (7) reported that 3 hours treatment with serum decreased the chemotactic response of NIH/3T3 cells to PDGF. Confluent cultures of human skin fibroblasts were growth arrested by incubation in serum-free MEM/2/mg/ml BSA for 3 days. Then the media were changed to MEM/I0% FBS. After 3, 8, 24, and 48 hours, their chemotactic responses to PDGF were determined. The chemotactic response to PDGF after exposure to FBS for 3 hours was markedly decreased (>50%), and the inhibitory effect of serum lasted for 24 hours. These results were similar to those of the previous report by Grotendorst (7).
Effect of Growth Factors on the Chemotadic Response Since serum is a mixture of many mitogens, we investigated whether any purified mitogens contained in serum could affect the chemotactic response of human skin fibroblasts to PDGF. Confluent cultures of human skin fibroblasts were incubated in serum-free MEM/BSA for 4 days. Then growth factors, including PDGF, FGF, EGF, and TGF-J3, were added separately to final concentrations of 20 ng/ml. After 3 and 24 hours, the . chemotactic responses to PDGF were tested. Three and 24 hours of preincubation with EGF showed inhibitory effects on the chemotactic response to
Fig. 2. Chemotaxis assay was performed and the filters were observed microscopically. 20 ng/ml of PDCF [PDGF (+)] or no PDGF [PDGF (-)] was added to the lower chamber.
PDGF. FGF also decreased the chemotactic response after 24 hours. The data presented here are consistent with the previous report that FGF and EGF inhibited the migratory response of NIH/3T3 cells to PDGF (7). Preincubation with TGF-J3 for 3 hours did not have any effect on the migratory response of human skin fibroblasts. However, 24 hours of preincubation with TGF-p markedly enhanced both the migration toward PDGF and the basal level of random migration in the control chamber without PDGF. The stimulatory effect of TGF-p on the cell migration may be attributed to enhanced cell attachment to the filter as a result of increased matrix synthesis induced by TGF-p. Discussion
In the process of wound healing, many kinds of biologically active substances are involved in a complex manner and considered to play key roles in the regulation of cells' proliferation, migration, and protein synthesis. Among those factors, PDGF is
656
Soma et al
one of the most important chemoattractants for connective tissue cells. The data presented here indicated that the migratory response to PDGF is regulated not only by the gradient of PDGF concentration, but also by other mitogens and the growth state of the cells. Grotendorst (7) reported that NIH/3T3 cells that are stimulated by mitogens show a decreased chemotactic response to PDGF and suggested that cells that are committed to entering the cell cycle lose their ability to migrate in response to PDGF. His observations were supported by our data presented here that human skin fibroblasts exposed to mitogens including EGF, FGF, and serum showed decreased chemotactic responses to PDGF. However, in sharp contrast to NIH/3T3 cells, human skin fibroblasts migrated to PDGF more when the cells were exponentially growing, suggesting that the chemotactic response of human skin fibroblasts may be regulated differently from that of fibroblasts from an immortal cell line. The observations that growing cells showed a high ability to migrate toward PDGF may account for the simultaneous stimulation of proliferation and migration of connective tissue cells in the early stages of wound healing.
References 1) Postlethwaite AE, Seyer LM, Kang AH: Chemotactic attraction of human fibroblasts to type I, II and III collagens and collagen-derived peptides, Proc Natl Acad Sci USA, 75: 871-875, 1978. 2) Gauss-Muller, Kleinman HK, Martin GR, Schiffman E: Role of attachment factors and attractants in fibroblast chemotaxis,] Lab Clin Med, 96: 1071-1080, 1980. 3) Postlethwaite AE, Keski-Oja], Moses HL, Kang AH: Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor-p,] &p Med, 165: 251-256, 1987. 4) Postlethwaite AE, Seyer LM: Fibroblast chemotaxis induction by human recombinant interleukin-4,] Clin Invest, 87: 2147-2152, 1991. 5) Seppa HE], Grotendorst GR, Seppa SI, Shiffman E, Martin GR: The platelet-derived growth factor is a chemoattractant for fibroblasts,] Cell Bioi, 92: 584588,1982. 6) Ross R, Raines EW, Bowen-Pope DF: The biology of platelet-derived growth factor, Cell, 46: 155-169,1986. 7) Grotendorst GR: Alteration of the chemotactic response ofNIH/3T3 cells to PDGFby growth factors, transformation, and tumor promoters, Cell, 36: 279285,1984. 8) Grotendorst GR, Seppa HE;J, Kleinman HK, Martin GR: Attachment of smooth muscle cells to collagen and their migration toward platelet-derived growth factor, Proc Natl &.ad Sci USA, 78: 3669-3672, 1981.
