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Thalidomide Increases Human Keratinocyte Migration and Proliferation
Thalidomide Increases Human Keratinocyte Migration and Proliferation Maria R. Nasca,1 Edel A. O’Toole,*1 Prema Palicharla,* Dennis P. West,* and David T. Woodley* Dermatology Clinic, University of Catania, Catania, Italy; *Department of Dermatology, Northwestern University, Chicago, Illinois, U.S.A.
Thalidomide is reported to have therapeutic utility in the treatment of pyoderma gangrenosum, Behc¸et’s disease, aphthous ulcers, and skin wounds. We investigated the effect of thalidomide on human keratinocyte proliferation and migration, two early and critical events in the re-epithelialization of skin wounds. Thalidomide at concentrations less than 1 µM did not affect keratinocyte viability. Using a thymidine incorporation assay, we found that thalidomide, at therapeutic concentrations, induced more than a 2.5-fold increase in the proliferative potential of the cells. Keratinocyte migration was assessed by two independent motility assays: a colloidal gold assay and an in vitro scratch assay. At optimal concentrations, thalidomide increased keratinocyte migration on a collagen matrix
more than 2-fold in the colloidal gold assay and more than 3-fold in the scratch assay over control. Although pro-migratory, thalidomide did not alter the level of metalloproteinase-9 secreted into culture medium. Thalidomide did, however, induce a 2–4-fold increase in keratinocyte-derived interleukin-8, a pro-migratory cellular autocrine factor. Human keratinocyte migration and proliferation are essential for re-epithelialization of skin wounds. Interleukin-8 increases human keratinocyte migration and proliferation and is chemotactic for keratinocytes. Therefore, thalidomide may modulate keratinocyte proliferation and motility by a chemokine-dependent pathway. Key words: cytokine/reepithelialization/wound healing. J Invest Dermatol 113:720– 724, 1999
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by demonstrating that it inhibited basic fibroblast growth factorinduced corneal neovascularization in rabbits (D’Amato et al, 1994). The effect of thalidomide on leukocytes has been extensively investigated in vitro; however, there are no studies in the literature regarding its effect on keratinocytes (Neubert et al, 1996). Thalidomide has been demonstrated to be of therapeutic benefit in a variety of mucocutaneous disorders including aphthous ulceration, Behc¸et’s disease, and pyoderma gangrenosum (Rustin et al, 1990; GardnerMedwin et al, 1994b; Jacobson et al, 1997; Hecker and Lebwohl, 1998). It has been assumed that the beneficial effect of thalidomide upon these conditions is due to the drug’s anti-inflammatory properties as it reduces polymorphonuclear leukocyte chemotaxis and phagocytosis, and also inhibits soluble inflammatory mediators such as prostaglandins (Nasca et al, 1999). Another common denominator to many conditions in which thalidomide has a therapeutic benefit, however, appears to be ulcerative skin wounds. Because, the healing of cutaneous ulceration requires re-epithelialization, we wondered if one of the beneficial effects of thalidomide might be directly upon keratinocyte motility and proliferation, two early and critical events in resurfacing skin wounds. A few hours after wounding, keratinocytes migrate laterally over the wound bed. Approximately 1 d later, the cells at the edge of the epithelium begin to proliferate and contribute cells to the advancing tongue of migrating keratinocytes involved in resurfacing the wound (Odland and Ross, 1968; Woodley et al, 1993). We therefore hypothesized that thalidomide might increase keratinocyte migration and/or proliferation, as these are two fundamental steps in reepithelialization of healing wounds. In this study, we used two independent in vitro migration assays to examine the effects of thalidomide on human keratinocyte migration on type I collagen, a matrix molecule found in the wound bed. We also examined the effect of thalidomide on the proliferative potential of human keratinocytes.
halidomide [(N)-phthalimidoglutarimide] is a synthetic derivative of glutamic acid. It was widely prescribed in the late 1950s as a sedative-hypnotic, but was withdrawn from the market in the early 1960s because it was found to be a potent teratogen (Ochonisky and Revuz, 1994; Tseng et al, 1996). Sheskin (1965, 1980) fortuitously found that it was extremely effective in the treatment of erythema nodosum leprosum. Subsequently, several other dermatoses have also been reported to be responsive to thalidomide. Consequently, there has been a growing interest in this drug, whose mechanism of action is not yet fully known or understood (Grosshans and Illy, 1984; Naafs and Faber, 1985; Gardner-Medwin and Powell, 1994a). Thalidomide has anti-angiogenic, anti-inflammatory, and immunomodulating properties. It inhibits leukocyte chemotaxis and phagocytosis, lysosome degranulation, and mast cell activation (Barnhill et al, 1984). It also induces a decrease in the T helper/T suppressor ratio of T cells (Gad et al, 1985; Moncada et al, 1985; Shannon et al, 1992; Nogueira et al, 1994). The immunologic effects of thalidomide may depend on the inhibition of tumor necrosis factor-α (Sampaio et al, 1991; Moreira et al, 1993), and upon the downregulation of several cell surface adhesion markers on blood leukocytes (Neubert et al, 1992, 1993; Nogueira et al, 1994). The anti-angiogenic effect of thalidomide was confirmed Manuscript received July 16, 1998; revised May 20, 1999; accepted for publication July 15, 1999. Reprint requests to: Dr. David T. Woodley, Division of Dermatology, University of Southern California, Ambulatory Health Center, 1355 San Pablo Street, Los Angeles, CA 90033, U.S.A. 1M.R. Nasca and E.A. O’Toole contributed equally to this work Abbreviation: MMP, metalloproteinase.
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0022-202X/99/$14.00 Copyright © 1999 by The Society for Investigative Dermatology, Inc.
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MATERIALS AND METHODS Materials Rat-tail collagen I was obtained from Collaborative Biochemicals. Trypan blue 0.4% was obtained from Sigma (St Louis, MO). Safety Solve was obtained from Research Products International (Mount Prospect, IL). Tritiated thymidine was obtained from ICN Biomedicals. Thalidomide (a racemic mixture of D[1] and L[–] forms was supplied by Pediatric Pharmaceuticals Incorporated (Westfield, NJ). Thalidomide was initially solubilized in dimethylsulfoxide and further dilutions were made in keratinocyte serum-free medium. Mitomycin-C was obtained from Sigma. Cell culture Human keratinocytes obtained from neonatal foreskins were cultured in low calcium (0.09 mM), serum-free MCDB 153 medium (Gibco BRL, Gaithersburg, MD) as described by Boyce and Ham (1983), and modified by O’Keefe and Chiu (1988). All experiments were performed using human keratinocytes between second and fourth passages. Keratinocytes from 10 donor foreskins were used in this study. At least three different donor foreskins were used in each experiment. Cell viability Lactate dehydrogenase cytotoxicity assay To quantitate the cellular toxicity of thalidomide, we used a commercially available Cytotoxicity Detection Kit from Boehringer-Mannheim. This is a colorimetric assay which measures the lactate dehydrogenase activity released from damaged cells. Keratinocytes were plated in 24 well plates (5000 cells per well), allowed to attach and then treated with increasing concentrations of thalidomide for 16 h. The medium was harvested and the assay was performed as described (Racher et al, 1990). Percentage cytotoxicity was calculated from the mean of triplicate absorbance values using a formula incorporating the experimental value, low control and high control. Cell migration assays Colloidal gold assay The method of Albrecht-Buehler (1977), as modified by Woodley et al (1988), was used to evaluate keratinocyte motility. Coverslips were coated with colloidal gold salts, washed, and type I collagen (8 µg per ml) in Hanks’ balanced salt solution supplemented with calcium and magnesium was added to each well. Dishes were incubated at 37°C for 2 h to permit immobilization of collagen to the coverslips. Passage 3 keratinocytes were plated in each well at a density of 750 cells per cm2. After attachment, media containing various concentrations of thalidomide were added to each well (0.001–100 nM). Triplicate wells were used for each experimental condition. Cultures were incubated at 37°C, 5% CO2 for 16 h, washed and fixed in 0.1% formaldehyde in phosphate-buffered saline. Because the doubling time of keratinocytes under these conditions is approximately 30 h, the assay assesses cellular motility unconfounded by cell division. To quantitate migration, cells were examined with an Olympus inverted microscope under dark-field optics and the percentage of each field occupied by migration tracks, a so-called migration index, was calculated using computer-assisted image analysis (Chen et al, 1993). The images of 10 fields per condition were analyzed.
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experimental condition. Each experiment was repeated three times and the counts were averaged. Gelatin zymography Keratinocytes were plated on tissue-culture plastic at a density of 20,000 cells per cm2. After the cells attached, unattached cells were aspirated and thalidomide was added at various concentrations (0.001–100 nM). Media were harvested after 12 h, centrifuged at 4°C (1500 3 g) for 10 min to remove cells and debris, and concentrated 5-fold with a Centricon 30 (Amicon, Beverly, MA). The effect of thalidomide on metalloproteinase-9 (MMP-9) secreted into the media was evaluated by gelatin-ladened zymography as previously described (Sarret et al, 1992a). Triplicate experiments were performed. Interleukin (IL)-8 protein determination Human keratinocytes were cultured in basal medium prior to trypsinization, then plated on tissue culture plastic at a concentration of 15,000 cells per cm2. After the cells attached, increasing concentrations of thalidomide were added to the dishes (0.001–100 nM), and the cells were incubated for 12 h. The concentration of IL-8 in the conditioned medium was then measured using a commercially available enzyme-linked immunosorbent assay (Quantikine; R&D Systems, Minneapolis, MN) able to detect 4.7 pg IL-8 per ml. Standard curves for recombinant human IL-8 ranging up to 2000 pg per ml were run for each assay. Protein levels in the supernatants were normalized to total cell number. Each supernatant was analyzed in triplicate. This experiment was repeated three times.
RESULTS Concentrations of thalidomide , 1 µM do not affect keratinocyte viability Keratinocyte viability after exposure to thalidomide was examined by calculating the percentage of nonviable cells using an lactate dehydrogenase release cytotoxicity assay. As shown in Fig 1, increasing cytotoxicity was observed with increasing concentrations of the drug, whereas concentrations , 1 µM minimally affected cell viability. Because keratinocyte cytotoxicity was observed at concentrations of thalidomide . 1 µM (Fig 1), we used thalidomide at concentrations between 0.001 and 100 nM for all biologic assays in this study. Thalidomide increases keratinocyte migration in vitro Human keratinocytes were exposed to a substratum of type I collagen (8 µg per ml). The keratinocytes are maximally stimulated to migrate on a type I collagen matrix of 15 µg per ml or greater. This optimal matrix provides migration indices above
In vitro scratch assay A second migration assay, the so-called ‘‘scratch assay’’ was performed according to Cha et al (1996). Keratinocytes (40,000 per cm2) were plated in tissue culture dishes, and after 24 h of incubation, the cells were incubated with 10 µg per ml of mitomycin-C for 2 h to inhibit cell proliferation, and the confluent monolayer of cells was scratched in a standardized manner with a plastic apparatus to create a cell-free zone in each well, 2 mm in width. The medium was aspirated and replaced with keratinocyte medium with or without the presence of thalidomide (0.001– 100 nM). The cells were then incubated for 24 h at 37°C. In vitro reepithelialization was documented by photography, and the amount of migration was quantitated by computer-assisted image analysis using NIH Image 1.6. The residual gap between the migrating keratinocytes was measured and expressed as a percentage of the area of the scratch remaining unfilled. Each experiment was performed in triplicate. Thymidine incorporation assay The proliferative potential of human keratinocyte cultures with and without the presence of thalidomide was determined by the thymidine incorporation assay of O’Keefe and Chiu (1988). Keratinocytes were plated in 24 well dishes (20,000 cells per cm2) and allowed to attach. Cells were then exposed to thalidomide diluted in keratinocyte medium (0.001–100 nM) for 12 h and 2 µCi of (3H)thymidine (New England Nuclear Products, Boston, MA) were added per well. Cells were incubated at 37°C for 8 h. Medium was aspirated and the cells were washed three times with ice-cold phosphate-buffered saline, twice with ice-cold trichloracetic acid and solubilized with 0.1 M NaOH containing 1% sodium dodecyl sulfate. Solubilized radioactivity was counted in a scintillation counter with Safety Solve. Ten wells were used for each
Figure 1. Concentrations of thalidomide , 1 µM do not affect keratinocyte viability. Keratinocytes were plated in 12 well plates, allowed to attach and treated with increasing concentrations of thalidomide. The medium was harvested and the amount of lactate dehydrogenase released into the medium calculated as described in the Materials and Methods. Data are expressed as the mean 6 SD of triplicate values from three experiments. *p , 0.05 compared with no added thalidomide. **p , 0.001 compared with no added thalidomide.
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30. Therefore, the 8 µg per ml collagen matrix used in these experiments is a ‘‘suboptimal’’ matrix that provides migration indices of approximately 20. As shown in Fig 2, the presence of thalidomide at 0.01 nM, 0.1 nM, and 1 nM increased the migration indices from 20.2 6 1.1 to 22.25 6 1.1 and 27.45 6 1.3, respectively. Triplicate independent experiments were performed. Thalidomide effect on keratinocyte motility with suboptimal amounts of collagen was statistically significant (p , 0.05), when analyzed by the Student’s t test. On an ‘‘optimal’’ matrix of 15 µg per ml of collagen, keratinocytes migrated maximally and no thalidomide enhancement in cellular motility could be detected (data not shown). To confirm the results of the colloidal gold assay, we assessed motility in the scratch assay with and without thalidomide. In the scratch assay, keratinocytes were plated densely in tissue culture wells for 24 h incubated with mitomycin-C to inhibit proliferation, then a standardized scratch was made through the confluent monolayer, as previously described (Cha et al, 1996). Keratinocytes from the cut edge of the scratch were then allowed to migrate for 12 h in the presence of increasing amounts of thalidomide and photographed. The residual scratch area between inwardly migrating keratinocytes from the edges of the scratch was measured as described in Materials and Methods. As shown in Fig 3, the keratinocytes migrated inwardly and covered a greater area of the scratch in the presence of thalidomide (compare 3B with 3G), when compared with assays without thalidomide (3A). As shown in the bar graph (Fig 3), keratinocytes on plastic exposed to thalidomide at concentrations of 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM, migrated to fill 42 6 3.9, 52 6 4.8, 61 6 5.9, 70 6 7.2, 82 6 7 and 90 6 9.2% of the scratch area, respectively. Concentrations greater than 0.1 nM significantly promoted keratinocyte migration (p , 0.05, Student’s t test). Thalidomide increases keratinocyte proliferation in vitro In order to determine if thalidomide affects the growth potential of keratinocyte cultures, the ability of human keratinocytes to incorporate tritiated thymidine in the presence of noncytotoxic doses of thalidomide was assessed according to O’Keefe and Chiu (1988). As shown in Fig 4, human keratinocytes cultured in serumfree medium in the presence of 0.01–100 nM of thalidomide had increased levels of thymidine incorporation compared with those without thalidomide. This effect was concentration-dependent up to a concentration of 0.1 nM. At concentrations . 0.1 nM, thalidomide increased keratinocyte proliferation, but not as significantly as with lower concentrations.
Figure 2. Thalidomide promotes keratinocyte migration in the colloidal gold assay. Keratinocytes were allowed to migrate for 16 h, in the presence of increasing concentrations of thalidomide. Ten migration indices were calculated for each condition and the experiment was repeated three times. Error bars; mean 6 SD. *p , 0.004 compared with no added thalidomide.
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Thalidomide does not alter keratinocyte gelatinase secretion Many factors that promote keratinocyte motility also increase MMP-9 activity, for example, hypoxia, collagen I, and cyclic adenosine monophosphate (Iwasaki et al, 1994; Sarret et al, 1992a; O’Toole et al, 1997). MMP-9 is also known as type IV collagenase/gelatinase and has a molecular weight of 92 kDa. Using standard gelatin zymography, we examined the expression of MMP-9 in keratinocytes cultured with and without nontoxic concentrations of thalidomide. Increasing concentrations of thalidomide did not alter keratinocyte expression of MMP-9 (data not shown). Thalidomide increases keratinocyte IL-8 production IL-8 serves as a keratinocyte autocrine factor and is a cytokine that promotes keratinocyte motility. We wished to determine if the thalidomide-induced enhancement of keratinocyte motility was associated with an altered expression of keratinocyte-derived IL-8. IL-8 production in the supernatant of keratinocytes with and without thalidomide was measured by enzyme-linked immunosorbent assay. Cell counts were performed to ensure that an approximately equal number of cells attached for each condition. IL8 protein activity was detected in unconcentrated conditioned keratinocyte medium. As shown in Fig 5, the presence of thalidomide increased IL-8 secretion in a concentration-dependent manner. Untreated keratinocytes, secreted basal levels of IL-8 (4.2 6 0.2 pg per ml). In the presence of 0.1 nM, 1 nM, and 10 nM of thalidomide, the keratinocytes secreted 9.2 6 0.9 pg IL-8 per ml, 11.5 6 0.8 pg IL-8 per ml, and 16.3 6 1 pg IL-8 per ml, respectively. DISCUSSION In recent years, thalidomide has been used successfully in the treatment of several dermatoses characterized by loss of tissue, including aphthous stomatitis, genital aphthosis, Behc¸et’s disease (Grinspan et al, 1989; Revuz et al, 1990; Revuz, 1990; GardnerMedwin et al, 1994b), aphthous ulcers in AIDS patients (Paterson et al, 1995; Jacobson et al, 1997), and pyoderma gangrenosum (Munro and Cox, 1988; Rustin et al, 1990). Thalidomide has a number of side-effects but most of these are relatively uncommon. Two of the most serious side-effects are peripheral neuropathy, which is not uncommon and often dose-related, and its very high incidence of teratogenicity in the event of fetal exposure. To insure that the effects of thalidomide upon keratinocyte migration and proliferation were not due to nonspecific toxicity of the drug, we determined the range at which thalidomide could be used without causing nonspecific toxicity using a lactate dehydrogenase cytotoxicity assay. The noncytotoxic concentrations of thalidomide used in the proliferation and motility assays were well within the therapeutically effective plasma levels of thalidomide used in human diseases (Chen et al, 1989; Boughton et al, 1995). The mode of action of thalidomide is unclear, but thalidomide is a potent immunomodulatory and anti-inflammatory agent (Paterson et al, 1995) that selectively reduces tumor necrosis factor-α expression (Sampaio et al, 1991). Tumor necrosis factor-α promotes neutrophil maturation and keratinocyte IL-8 secretion, which enhances neutrophil chemotaxis (Moreira et al, 1993; Costa et al, 1994). Neutrophils at the wound site destroy contaminating bacteria (Tannesh et al, 1988), but this may cause further damage to healing tissue. Keratinocyte migration and proliferation are fundamentally important steps in re-epithelializing skin wounds (Woodley et al, 1993). In this study, thalidomide not only promoted keratinocyte motility in two independent migration assays, but also increased the cell’s proliferative potential. Thus, thalidomide may influence two fundamental mechanisms involved in wound healing. In this study, low concentrations of thalidomide produced a marked increase in keratinocyte proliferation; however, higher concentrations were required to increase keratinocyte migration significantly. This may be because keratinocyte migration requires new protein synthesis, redistribution of lamellipodia-associated proteins, and synthesis and secretion of collagenases (O’Toole et al, 1997). These
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Figure 3. Thalidomide promotes keratinocyte migration in the in vitro scratch assay. An in vitro wound was introduced in confluent cultures of normal human keratinocytes, as described in the Materials and Methods. The cells were incubated without thalidomide (A) or with increasing concentrations of thalidomide (B–G). After 24 h, the cells were photographed under phase-contrast microscopy. Scale bar: 250 µm. The percentage of the original scratch area remaining unfilled was calculated by image analysis and expressed as the mean 6 SD of three observations (graph). 10–12–10–7 5 0.001–100 nM.
Figure 4. Thalidomide promotes keratinocyte proliferation in vitro. Subconfluent keratinocytes in 24 well plates were exposed to increasing concentrations of thalidomide for 12 h, then incubated with 2 µCi of thymidine for an additional 8 h. Cellular samples were prepared as described in the Materials and Methods and the amount of solubilized radioactivity was counted in a scintillation counter. Error bars, mean 6 SD of three experiments performed in triplicate. **p , 0.0005 compared with control without thalidomide. *p , 0.001 compared with control without thalidomide.
processes may require higher doses of thalidomide compared with DNA synthesis. Matrix-induced promotion of keratinocyte motility is associated with increased synthesis of collagenases (Petersen et al, 1987, 1989; Sarret et al, 1992a; Iwasaki et al, 1994). Degradation of matrix allows for detachment of keratinocyte-matrix adhesions, and this process allows the cell to continue to migrate during re-epithelialization. Our study demonstrates that thalidomide does not alter the expression of keratinocyte-derived MMP-9, a collagenase linked to keratinocyte migration on a collagen matrix (Sarret et al, 1992a). Therefore, the enhancement of human keratinocyte motility by thalidomide likely involves some other mechanism.
Figure 5. Thalidomide increases human keratinocyte IL-8 secretion. Keratinocytes cultured in basal medium were plated on plastic at a concentration of 15,000 cells per cm2. After cell attachment, increasing concentrations of thalidomide were added to the dishes. After 12 h, the concentration of IL-8 was measured in unconcentrated conditioned medium. Error bars, mean 6 SD of three experiments performed in triplicate. *p , 0.001 compared with control without thalidomide. **p , 0.0005 compared with control without thalidomide.
IL-8 is known to enhance human keratinocyte chemotaxis (Schro¨der, 1992) and proliferation (Tuschil et al, 1992) and to be increased in diseases characterized by epidermal hyperproliferation, such as psoriasis (Nickoloff et al, 1991, 1994). In this study, low concentrations of thalidomide (0.01–0.1 nM) promoted human keratinocyte proliferation, whereas higher concentrations also promoted human keratinocyte proliferation, but not as markedly as the lower concentrations. These data are in accordance with other studies of other cell types showing opposite biologic effects depending upon the concentration of thalidomide used (McHugh et al, 1995). IL-8 secretion, evaluated by enzyme-linked immunosorbent assay, showed a 2–4-fold increase compared with control human keratinocytes in the presence of thalidomide. Therefore,
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the thalidomide-induced enhancement of keratinocyte motility may be mediated via an increased expression of IL-8 which would act as a pro-migratory autocrine factor. Keratinocyte migration and proliferation are independent mechanisms (Sarret et al, 1992b; Woodley et al, 1993; Iwasaki et al, 1994). The timing of the proliferative and locomotion effects during wound healing are different, as the enhancement of keratinocyte migration occurs early, whereas the increase in cell division takes place later (Woodley et al, 1993). Moreover, keratinocyte proliferation may be enhanced by soluble factors and cytokines that have no effect, or, alternatively, have an inhibitory action on keratinocyte motility (Sarret et al, 1992b). Epidermal growth factor and transforming growth factor-α enhance both keratinocyte migration and proliferation (Cha et al, 1996), but these effects take place by independent mechanisms (Chen et al, 1993). In this study, we find that thalidomide enhances both keratinocyte migration and proliferation in vitro. The therapeutic value of thalidomide in the treatment of chronic wounds has yet to be determined. The positive effect of thalidomide on both migration and proliferation in vitro, and the many anecdotal reports of successful treatment of chronic ulcerating conditions with thalidomide suggest that it may be of value in conditions where there is deficient re-epithelialization.
This study was supported by Northwestern Memorial Foundation (DPW), a Howard Hughes Medical Institute Physician Fellowship (EOT), and Grants P01 AR41045, R01 AR46538, and R01 AR33625 from the National Institute of Health, Bethesda, MD (DTW). Maria R. Nasca carried out this work as a visiting Assistant Professor at North-western University.
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Simultaneous up- and down-regulation of different integrin receptors on human white blood cells. Life Sci 55:77–92, 1994 O’Keefe EJ, Chiu ML: Stimulation of thymidine incorporation in keratinocytes by insulin, epidermal growth factor, and placental extract: comparison with cell number to assess growth. J Invest Dermatol 90:2–7, 1988 O’Toole EA, Marinkovich MP, Peavey CL, Amieva MR, Furthmayr H, Mustor TA, Woodley DT: Hypoxia increases human keratinocyte motility on connective tissue. J Clin Invest 100:2881–2891, 1997 Ochonisky S, Revuz J: Thalidomide use in dermatology. Eur J Dermatol 4:9–15, 1994 Odland G, Ross R: Human wound repair. I. Epidermal regeneration. J Cell Biol 39:135– 151, 1968 Paterson DL, Georghiou PR, Allworth AM, Kemp RJ: Thalidomide as treatment of refractory aphthous ulceration related to human immunodeficiency virus infection. Clin Infect Dis 20:250–254, 1995 Petersen MJ, Woodley DT, Stricklin GP, O’Keefe EJ: Production of procollagenase by cultured human keratinocytes. J Biol Chem 262:835–840, 1987 Petersen MJ, Woodley DT, Stricklin GP, O’Keefe EJ: Constitutive production of procollagenase and tissue inhibitor of metalloproteinases by human keratinocytes in culture. J Invest Dermatol 92:156–159, 1989 Racher AJ, Looby D, Griffiths JB: Use of lactate dehydrogenase release to assess changes in culture viability. Cytotechnology 3:301–307, 1990 Revuz J: Actualite´ du thalidomide. Ann Dermatol Venereol 117:313–321, 1990 Revuz J, Guillaume J-C, Janier M, et al: Crossover study of thalidomide vs placebo in severe recurrent aphthous stomatitis. Arch Dermatol 126:923–927, 1990 Rustin MHA, Gilkes JJH, Robinson TWE: Pyoderma gangrenosum associated with Behc¸et’s disease: treatment with thalidomide. J Am Acad Dermatol 23:941–944, 1990 Sampaio EP, Sarno EN, Galilly R, Cohn ZA, Kaplan G: Thalidomide selectively inhibits tumor necrosis factor α production by stimulated human monocytes. J Exp Med 173:699–703, 1991 Sarret Y, Woodley DT, Goldberg GS, Kronberger A, Wynn KC: Constitutive synthesis of a 92-kDa keratinocyte-derived type IV collagenase is enhanced by type I collagen and decreased by type IV collagen matrices. J Invest Dermatol 99:836– 841, 1992a Sarret Y, Woodley DT, Grigsby K, Wynn K, O’Keefe EJ: Human keratinocyte locomotion: the effect of selected cytokines. J Invest Dermatol 98:400–405, 1992b Schro¨der JM: Chemotactic cytokines in the epidermis. Exp Dermatol 1:12–19, 1992 Shannon EJ, Ejigu M, Haile-Mariam HS, Berhan TY, Tasesse G: Thalidomide’s effectiveness in erythema nodosum leprosum is associated with a decrease in CD41 cells in the peripheral blood. Lepr Rev 63:5–11, 1992 Sheskin J: Thalidomide in the treatment of lepra reaction. Clin Pharmacol Ther 6:303– 306, 1965 Sheskin J: The treatment of lepra reaction in lepromatous leprosy: fifteen years’ experience with thalidomide. Int J Dermatol 19:318–322, 1980 Tannesh MG, Worther GS, Johnston RBJ. Neutrophil emigration activation and tissue damage. In: Clark RAF, Hanson PM (eds). Molecular and Cellular Biology of Wound Repair. New York: Plenum Press, 1988 Tseng S, Pak G, Washenik K, Keltz Pomeranz M, Shupack JL: Rediscovering thalidomide: a review of its mechanism of action, side effects and potential uses. J Am Acad Dermatol 35:969–979, 1996 Tuschil A, Lam C, Haslberger A, Lindley I: Interleukin-8 stimulates calcium transients and promotes epidermal cell proliferation. J Invest Dermatol 99:294–298, 1992 Woodley DT, Bachmann PM, O’Keefe EJ: Laminin inhibits human keratinocyte migration. J Cell Physiol 136:140–146, 1988 Woodley DT, Chen JD, Kim JP, Sarret Y, Iwasaki T, Kim YH, O’Keefe EJ: Re-epithelialization. Human keratinocyte locomotion. Dermatol Clin 11:641–646, 1993
Thalidomide is reported to have therapeutic utility in the treatment of pyoderma gangrenosum, Behc¸et’s disease, aphthous ulcers, and skin wounds. We investigated the effect of thalidomide on human keratinocyte proliferation and migration, two early and critical events in the re-epithelialization of skin wounds. Thalidomide at concentrations less than 1 µM did not affect keratinocyte viability. Using a thymidine incorporation assay, we found that thalidomide, at therapeutic concentrations, induced more than a 2.5-fold increase in the proliferative potential of the cells. Keratinocyte migration was assessed by two independent motility assays: a colloidal gold assay and an in vitro scratch assay. At optimal concentrations, thalidomide increased keratinocyte migration on a collagen matrix
more than 2-fold in the colloidal gold assay and more than 3-fold in the scratch assay over control. Although pro-migratory, thalidomide did not alter the level of metalloproteinase-9 secreted into culture medium. Thalidomide did, however, induce a 2–4-fold increase in keratinocyte-derived interleukin-8, a pro-migratory cellular autocrine factor. Human keratinocyte migration and proliferation are essential for re-epithelialization of skin wounds. Interleukin-8 increases human keratinocyte migration and proliferation and is chemotactic for keratinocytes. Therefore, thalidomide may modulate keratinocyte proliferation and motility by a chemokine-dependent pathway. Key words: cytokine/reepithelialization/wound healing. J Invest Dermatol 113:720– 724, 1999
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by demonstrating that it inhibited basic fibroblast growth factorinduced corneal neovascularization in rabbits (D’Amato et al, 1994). The effect of thalidomide on leukocytes has been extensively investigated in vitro; however, there are no studies in the literature regarding its effect on keratinocytes (Neubert et al, 1996). Thalidomide has been demonstrated to be of therapeutic benefit in a variety of mucocutaneous disorders including aphthous ulceration, Behc¸et’s disease, and pyoderma gangrenosum (Rustin et al, 1990; GardnerMedwin et al, 1994b; Jacobson et al, 1997; Hecker and Lebwohl, 1998). It has been assumed that the beneficial effect of thalidomide upon these conditions is due to the drug’s anti-inflammatory properties as it reduces polymorphonuclear leukocyte chemotaxis and phagocytosis, and also inhibits soluble inflammatory mediators such as prostaglandins (Nasca et al, 1999). Another common denominator to many conditions in which thalidomide has a therapeutic benefit, however, appears to be ulcerative skin wounds. Because, the healing of cutaneous ulceration requires re-epithelialization, we wondered if one of the beneficial effects of thalidomide might be directly upon keratinocyte motility and proliferation, two early and critical events in resurfacing skin wounds. A few hours after wounding, keratinocytes migrate laterally over the wound bed. Approximately 1 d later, the cells at the edge of the epithelium begin to proliferate and contribute cells to the advancing tongue of migrating keratinocytes involved in resurfacing the wound (Odland and Ross, 1968; Woodley et al, 1993). We therefore hypothesized that thalidomide might increase keratinocyte migration and/or proliferation, as these are two fundamental steps in reepithelialization of healing wounds. In this study, we used two independent in vitro migration assays to examine the effects of thalidomide on human keratinocyte migration on type I collagen, a matrix molecule found in the wound bed. We also examined the effect of thalidomide on the proliferative potential of human keratinocytes.
halidomide [(N)-phthalimidoglutarimide] is a synthetic derivative of glutamic acid. It was widely prescribed in the late 1950s as a sedative-hypnotic, but was withdrawn from the market in the early 1960s because it was found to be a potent teratogen (Ochonisky and Revuz, 1994; Tseng et al, 1996). Sheskin (1965, 1980) fortuitously found that it was extremely effective in the treatment of erythema nodosum leprosum. Subsequently, several other dermatoses have also been reported to be responsive to thalidomide. Consequently, there has been a growing interest in this drug, whose mechanism of action is not yet fully known or understood (Grosshans and Illy, 1984; Naafs and Faber, 1985; Gardner-Medwin and Powell, 1994a). Thalidomide has anti-angiogenic, anti-inflammatory, and immunomodulating properties. It inhibits leukocyte chemotaxis and phagocytosis, lysosome degranulation, and mast cell activation (Barnhill et al, 1984). It also induces a decrease in the T helper/T suppressor ratio of T cells (Gad et al, 1985; Moncada et al, 1985; Shannon et al, 1992; Nogueira et al, 1994). The immunologic effects of thalidomide may depend on the inhibition of tumor necrosis factor-α (Sampaio et al, 1991; Moreira et al, 1993), and upon the downregulation of several cell surface adhesion markers on blood leukocytes (Neubert et al, 1992, 1993; Nogueira et al, 1994). The anti-angiogenic effect of thalidomide was confirmed Manuscript received July 16, 1998; revised May 20, 1999; accepted for publication July 15, 1999. Reprint requests to: Dr. David T. Woodley, Division of Dermatology, University of Southern California, Ambulatory Health Center, 1355 San Pablo Street, Los Angeles, CA 90033, U.S.A. 1M.R. Nasca and E.A. O’Toole contributed equally to this work Abbreviation: MMP, metalloproteinase.
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MATERIALS AND METHODS Materials Rat-tail collagen I was obtained from Collaborative Biochemicals. Trypan blue 0.4% was obtained from Sigma (St Louis, MO). Safety Solve was obtained from Research Products International (Mount Prospect, IL). Tritiated thymidine was obtained from ICN Biomedicals. Thalidomide (a racemic mixture of D[1] and L[–] forms was supplied by Pediatric Pharmaceuticals Incorporated (Westfield, NJ). Thalidomide was initially solubilized in dimethylsulfoxide and further dilutions were made in keratinocyte serum-free medium. Mitomycin-C was obtained from Sigma. Cell culture Human keratinocytes obtained from neonatal foreskins were cultured in low calcium (0.09 mM), serum-free MCDB 153 medium (Gibco BRL, Gaithersburg, MD) as described by Boyce and Ham (1983), and modified by O’Keefe and Chiu (1988). All experiments were performed using human keratinocytes between second and fourth passages. Keratinocytes from 10 donor foreskins were used in this study. At least three different donor foreskins were used in each experiment. Cell viability Lactate dehydrogenase cytotoxicity assay To quantitate the cellular toxicity of thalidomide, we used a commercially available Cytotoxicity Detection Kit from Boehringer-Mannheim. This is a colorimetric assay which measures the lactate dehydrogenase activity released from damaged cells. Keratinocytes were plated in 24 well plates (5000 cells per well), allowed to attach and then treated with increasing concentrations of thalidomide for 16 h. The medium was harvested and the assay was performed as described (Racher et al, 1990). Percentage cytotoxicity was calculated from the mean of triplicate absorbance values using a formula incorporating the experimental value, low control and high control. Cell migration assays Colloidal gold assay The method of Albrecht-Buehler (1977), as modified by Woodley et al (1988), was used to evaluate keratinocyte motility. Coverslips were coated with colloidal gold salts, washed, and type I collagen (8 µg per ml) in Hanks’ balanced salt solution supplemented with calcium and magnesium was added to each well. Dishes were incubated at 37°C for 2 h to permit immobilization of collagen to the coverslips. Passage 3 keratinocytes were plated in each well at a density of 750 cells per cm2. After attachment, media containing various concentrations of thalidomide were added to each well (0.001–100 nM). Triplicate wells were used for each experimental condition. Cultures were incubated at 37°C, 5% CO2 for 16 h, washed and fixed in 0.1% formaldehyde in phosphate-buffered saline. Because the doubling time of keratinocytes under these conditions is approximately 30 h, the assay assesses cellular motility unconfounded by cell division. To quantitate migration, cells were examined with an Olympus inverted microscope under dark-field optics and the percentage of each field occupied by migration tracks, a so-called migration index, was calculated using computer-assisted image analysis (Chen et al, 1993). The images of 10 fields per condition were analyzed.
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experimental condition. Each experiment was repeated three times and the counts were averaged. Gelatin zymography Keratinocytes were plated on tissue-culture plastic at a density of 20,000 cells per cm2. After the cells attached, unattached cells were aspirated and thalidomide was added at various concentrations (0.001–100 nM). Media were harvested after 12 h, centrifuged at 4°C (1500 3 g) for 10 min to remove cells and debris, and concentrated 5-fold with a Centricon 30 (Amicon, Beverly, MA). The effect of thalidomide on metalloproteinase-9 (MMP-9) secreted into the media was evaluated by gelatin-ladened zymography as previously described (Sarret et al, 1992a). Triplicate experiments were performed. Interleukin (IL)-8 protein determination Human keratinocytes were cultured in basal medium prior to trypsinization, then plated on tissue culture plastic at a concentration of 15,000 cells per cm2. After the cells attached, increasing concentrations of thalidomide were added to the dishes (0.001–100 nM), and the cells were incubated for 12 h. The concentration of IL-8 in the conditioned medium was then measured using a commercially available enzyme-linked immunosorbent assay (Quantikine; R&D Systems, Minneapolis, MN) able to detect 4.7 pg IL-8 per ml. Standard curves for recombinant human IL-8 ranging up to 2000 pg per ml were run for each assay. Protein levels in the supernatants were normalized to total cell number. Each supernatant was analyzed in triplicate. This experiment was repeated three times.
RESULTS Concentrations of thalidomide , 1 µM do not affect keratinocyte viability Keratinocyte viability after exposure to thalidomide was examined by calculating the percentage of nonviable cells using an lactate dehydrogenase release cytotoxicity assay. As shown in Fig 1, increasing cytotoxicity was observed with increasing concentrations of the drug, whereas concentrations , 1 µM minimally affected cell viability. Because keratinocyte cytotoxicity was observed at concentrations of thalidomide . 1 µM (Fig 1), we used thalidomide at concentrations between 0.001 and 100 nM for all biologic assays in this study. Thalidomide increases keratinocyte migration in vitro Human keratinocytes were exposed to a substratum of type I collagen (8 µg per ml). The keratinocytes are maximally stimulated to migrate on a type I collagen matrix of 15 µg per ml or greater. This optimal matrix provides migration indices above
In vitro scratch assay A second migration assay, the so-called ‘‘scratch assay’’ was performed according to Cha et al (1996). Keratinocytes (40,000 per cm2) were plated in tissue culture dishes, and after 24 h of incubation, the cells were incubated with 10 µg per ml of mitomycin-C for 2 h to inhibit cell proliferation, and the confluent monolayer of cells was scratched in a standardized manner with a plastic apparatus to create a cell-free zone in each well, 2 mm in width. The medium was aspirated and replaced with keratinocyte medium with or without the presence of thalidomide (0.001– 100 nM). The cells were then incubated for 24 h at 37°C. In vitro reepithelialization was documented by photography, and the amount of migration was quantitated by computer-assisted image analysis using NIH Image 1.6. The residual gap between the migrating keratinocytes was measured and expressed as a percentage of the area of the scratch remaining unfilled. Each experiment was performed in triplicate. Thymidine incorporation assay The proliferative potential of human keratinocyte cultures with and without the presence of thalidomide was determined by the thymidine incorporation assay of O’Keefe and Chiu (1988). Keratinocytes were plated in 24 well dishes (20,000 cells per cm2) and allowed to attach. Cells were then exposed to thalidomide diluted in keratinocyte medium (0.001–100 nM) for 12 h and 2 µCi of (3H)thymidine (New England Nuclear Products, Boston, MA) were added per well. Cells were incubated at 37°C for 8 h. Medium was aspirated and the cells were washed three times with ice-cold phosphate-buffered saline, twice with ice-cold trichloracetic acid and solubilized with 0.1 M NaOH containing 1% sodium dodecyl sulfate. Solubilized radioactivity was counted in a scintillation counter with Safety Solve. Ten wells were used for each
Figure 1. Concentrations of thalidomide , 1 µM do not affect keratinocyte viability. Keratinocytes were plated in 12 well plates, allowed to attach and treated with increasing concentrations of thalidomide. The medium was harvested and the amount of lactate dehydrogenase released into the medium calculated as described in the Materials and Methods. Data are expressed as the mean 6 SD of triplicate values from three experiments. *p , 0.05 compared with no added thalidomide. **p , 0.001 compared with no added thalidomide.
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30. Therefore, the 8 µg per ml collagen matrix used in these experiments is a ‘‘suboptimal’’ matrix that provides migration indices of approximately 20. As shown in Fig 2, the presence of thalidomide at 0.01 nM, 0.1 nM, and 1 nM increased the migration indices from 20.2 6 1.1 to 22.25 6 1.1 and 27.45 6 1.3, respectively. Triplicate independent experiments were performed. Thalidomide effect on keratinocyte motility with suboptimal amounts of collagen was statistically significant (p , 0.05), when analyzed by the Student’s t test. On an ‘‘optimal’’ matrix of 15 µg per ml of collagen, keratinocytes migrated maximally and no thalidomide enhancement in cellular motility could be detected (data not shown). To confirm the results of the colloidal gold assay, we assessed motility in the scratch assay with and without thalidomide. In the scratch assay, keratinocytes were plated densely in tissue culture wells for 24 h incubated with mitomycin-C to inhibit proliferation, then a standardized scratch was made through the confluent monolayer, as previously described (Cha et al, 1996). Keratinocytes from the cut edge of the scratch were then allowed to migrate for 12 h in the presence of increasing amounts of thalidomide and photographed. The residual scratch area between inwardly migrating keratinocytes from the edges of the scratch was measured as described in Materials and Methods. As shown in Fig 3, the keratinocytes migrated inwardly and covered a greater area of the scratch in the presence of thalidomide (compare 3B with 3G), when compared with assays without thalidomide (3A). As shown in the bar graph (Fig 3), keratinocytes on plastic exposed to thalidomide at concentrations of 0.001 nM, 0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM, migrated to fill 42 6 3.9, 52 6 4.8, 61 6 5.9, 70 6 7.2, 82 6 7 and 90 6 9.2% of the scratch area, respectively. Concentrations greater than 0.1 nM significantly promoted keratinocyte migration (p , 0.05, Student’s t test). Thalidomide increases keratinocyte proliferation in vitro In order to determine if thalidomide affects the growth potential of keratinocyte cultures, the ability of human keratinocytes to incorporate tritiated thymidine in the presence of noncytotoxic doses of thalidomide was assessed according to O’Keefe and Chiu (1988). As shown in Fig 4, human keratinocytes cultured in serumfree medium in the presence of 0.01–100 nM of thalidomide had increased levels of thymidine incorporation compared with those without thalidomide. This effect was concentration-dependent up to a concentration of 0.1 nM. At concentrations . 0.1 nM, thalidomide increased keratinocyte proliferation, but not as significantly as with lower concentrations.
Figure 2. Thalidomide promotes keratinocyte migration in the colloidal gold assay. Keratinocytes were allowed to migrate for 16 h, in the presence of increasing concentrations of thalidomide. Ten migration indices were calculated for each condition and the experiment was repeated three times. Error bars; mean 6 SD. *p , 0.004 compared with no added thalidomide.
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Thalidomide does not alter keratinocyte gelatinase secretion Many factors that promote keratinocyte motility also increase MMP-9 activity, for example, hypoxia, collagen I, and cyclic adenosine monophosphate (Iwasaki et al, 1994; Sarret et al, 1992a; O’Toole et al, 1997). MMP-9 is also known as type IV collagenase/gelatinase and has a molecular weight of 92 kDa. Using standard gelatin zymography, we examined the expression of MMP-9 in keratinocytes cultured with and without nontoxic concentrations of thalidomide. Increasing concentrations of thalidomide did not alter keratinocyte expression of MMP-9 (data not shown). Thalidomide increases keratinocyte IL-8 production IL-8 serves as a keratinocyte autocrine factor and is a cytokine that promotes keratinocyte motility. We wished to determine if the thalidomide-induced enhancement of keratinocyte motility was associated with an altered expression of keratinocyte-derived IL-8. IL-8 production in the supernatant of keratinocytes with and without thalidomide was measured by enzyme-linked immunosorbent assay. Cell counts were performed to ensure that an approximately equal number of cells attached for each condition. IL8 protein activity was detected in unconcentrated conditioned keratinocyte medium. As shown in Fig 5, the presence of thalidomide increased IL-8 secretion in a concentration-dependent manner. Untreated keratinocytes, secreted basal levels of IL-8 (4.2 6 0.2 pg per ml). In the presence of 0.1 nM, 1 nM, and 10 nM of thalidomide, the keratinocytes secreted 9.2 6 0.9 pg IL-8 per ml, 11.5 6 0.8 pg IL-8 per ml, and 16.3 6 1 pg IL-8 per ml, respectively. DISCUSSION In recent years, thalidomide has been used successfully in the treatment of several dermatoses characterized by loss of tissue, including aphthous stomatitis, genital aphthosis, Behc¸et’s disease (Grinspan et al, 1989; Revuz et al, 1990; Revuz, 1990; GardnerMedwin et al, 1994b), aphthous ulcers in AIDS patients (Paterson et al, 1995; Jacobson et al, 1997), and pyoderma gangrenosum (Munro and Cox, 1988; Rustin et al, 1990). Thalidomide has a number of side-effects but most of these are relatively uncommon. Two of the most serious side-effects are peripheral neuropathy, which is not uncommon and often dose-related, and its very high incidence of teratogenicity in the event of fetal exposure. To insure that the effects of thalidomide upon keratinocyte migration and proliferation were not due to nonspecific toxicity of the drug, we determined the range at which thalidomide could be used without causing nonspecific toxicity using a lactate dehydrogenase cytotoxicity assay. The noncytotoxic concentrations of thalidomide used in the proliferation and motility assays were well within the therapeutically effective plasma levels of thalidomide used in human diseases (Chen et al, 1989; Boughton et al, 1995). The mode of action of thalidomide is unclear, but thalidomide is a potent immunomodulatory and anti-inflammatory agent (Paterson et al, 1995) that selectively reduces tumor necrosis factor-α expression (Sampaio et al, 1991). Tumor necrosis factor-α promotes neutrophil maturation and keratinocyte IL-8 secretion, which enhances neutrophil chemotaxis (Moreira et al, 1993; Costa et al, 1994). Neutrophils at the wound site destroy contaminating bacteria (Tannesh et al, 1988), but this may cause further damage to healing tissue. Keratinocyte migration and proliferation are fundamentally important steps in re-epithelializing skin wounds (Woodley et al, 1993). In this study, thalidomide not only promoted keratinocyte motility in two independent migration assays, but also increased the cell’s proliferative potential. Thus, thalidomide may influence two fundamental mechanisms involved in wound healing. In this study, low concentrations of thalidomide produced a marked increase in keratinocyte proliferation; however, higher concentrations were required to increase keratinocyte migration significantly. This may be because keratinocyte migration requires new protein synthesis, redistribution of lamellipodia-associated proteins, and synthesis and secretion of collagenases (O’Toole et al, 1997). These
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Figure 3. Thalidomide promotes keratinocyte migration in the in vitro scratch assay. An in vitro wound was introduced in confluent cultures of normal human keratinocytes, as described in the Materials and Methods. The cells were incubated without thalidomide (A) or with increasing concentrations of thalidomide (B–G). After 24 h, the cells were photographed under phase-contrast microscopy. Scale bar: 250 µm. The percentage of the original scratch area remaining unfilled was calculated by image analysis and expressed as the mean 6 SD of three observations (graph). 10–12–10–7 5 0.001–100 nM.
Figure 4. Thalidomide promotes keratinocyte proliferation in vitro. Subconfluent keratinocytes in 24 well plates were exposed to increasing concentrations of thalidomide for 12 h, then incubated with 2 µCi of thymidine for an additional 8 h. Cellular samples were prepared as described in the Materials and Methods and the amount of solubilized radioactivity was counted in a scintillation counter. Error bars, mean 6 SD of three experiments performed in triplicate. **p , 0.0005 compared with control without thalidomide. *p , 0.001 compared with control without thalidomide.
processes may require higher doses of thalidomide compared with DNA synthesis. Matrix-induced promotion of keratinocyte motility is associated with increased synthesis of collagenases (Petersen et al, 1987, 1989; Sarret et al, 1992a; Iwasaki et al, 1994). Degradation of matrix allows for detachment of keratinocyte-matrix adhesions, and this process allows the cell to continue to migrate during re-epithelialization. Our study demonstrates that thalidomide does not alter the expression of keratinocyte-derived MMP-9, a collagenase linked to keratinocyte migration on a collagen matrix (Sarret et al, 1992a). Therefore, the enhancement of human keratinocyte motility by thalidomide likely involves some other mechanism.
Figure 5. Thalidomide increases human keratinocyte IL-8 secretion. Keratinocytes cultured in basal medium were plated on plastic at a concentration of 15,000 cells per cm2. After cell attachment, increasing concentrations of thalidomide were added to the dishes. After 12 h, the concentration of IL-8 was measured in unconcentrated conditioned medium. Error bars, mean 6 SD of three experiments performed in triplicate. *p , 0.001 compared with control without thalidomide. **p , 0.0005 compared with control without thalidomide.
IL-8 is known to enhance human keratinocyte chemotaxis (Schro¨der, 1992) and proliferation (Tuschil et al, 1992) and to be increased in diseases characterized by epidermal hyperproliferation, such as psoriasis (Nickoloff et al, 1991, 1994). In this study, low concentrations of thalidomide (0.01–0.1 nM) promoted human keratinocyte proliferation, whereas higher concentrations also promoted human keratinocyte proliferation, but not as markedly as the lower concentrations. These data are in accordance with other studies of other cell types showing opposite biologic effects depending upon the concentration of thalidomide used (McHugh et al, 1995). IL-8 secretion, evaluated by enzyme-linked immunosorbent assay, showed a 2–4-fold increase compared with control human keratinocytes in the presence of thalidomide. Therefore,
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the thalidomide-induced enhancement of keratinocyte motility may be mediated via an increased expression of IL-8 which would act as a pro-migratory autocrine factor. Keratinocyte migration and proliferation are independent mechanisms (Sarret et al, 1992b; Woodley et al, 1993; Iwasaki et al, 1994). The timing of the proliferative and locomotion effects during wound healing are different, as the enhancement of keratinocyte migration occurs early, whereas the increase in cell division takes place later (Woodley et al, 1993). Moreover, keratinocyte proliferation may be enhanced by soluble factors and cytokines that have no effect, or, alternatively, have an inhibitory action on keratinocyte motility (Sarret et al, 1992b). Epidermal growth factor and transforming growth factor-α enhance both keratinocyte migration and proliferation (Cha et al, 1996), but these effects take place by independent mechanisms (Chen et al, 1993). In this study, we find that thalidomide enhances both keratinocyte migration and proliferation in vitro. The therapeutic value of thalidomide in the treatment of chronic wounds has yet to be determined. The positive effect of thalidomide on both migration and proliferation in vitro, and the many anecdotal reports of successful treatment of chronic ulcerating conditions with thalidomide suggest that it may be of value in conditions where there is deficient re-epithelialization.
This study was supported by Northwestern Memorial Foundation (DPW), a Howard Hughes Medical Institute Physician Fellowship (EOT), and Grants P01 AR41045, R01 AR46538, and R01 AR33625 from the National Institute of Health, Bethesda, MD (DTW). Maria R. Nasca carried out this work as a visiting Assistant Professor at North-western University.
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