Interleukin 6 Indirectly Induces Keratinocyte Migration
2004; Elsevier BV; Volume: 122; Issue: 3 Linguagem: Inglês
10.1111/j.0022-202x.2004.22323.x
ISSN1523-1747
AutoresRandle M. Gallucci, Dusti K. Sloan, Julie M. Heck, Anne R. Murray, Sijy J. O’Dell,
Tópico(s)Cell Adhesion Molecules Research
ResumoIL-6-deficient transgenic mice (IL-6 KO) display significantly delayed cutaneous wound healing. To further elucidate the role of IL-6 in skin wound healing, epidermal keratinocyte and dermal fibroblast cells were isolated from neonatal IL-6 KO mice and treated with rmIL-6. It was found that rmIL-6 alone did not significantly modulate the proliferation or migration of cultured IL-6 KO keratinocytes. rmIL-6, however, significantly induced the migration of IL-6 KO keratinocytes (up to 5-fold) when co-cultured with dermal fibroblasts. Culture supernatants from IL-6-treated fibroblasts were also found to induce the migration of keratinocytes to a similar degree. Genomics analysis of treated fibroblasts indicated that rmIL-6 does not induce any known soluble keratinocyte migratory factors. rmIL-6 treatment of fibroblast, however, induced a rapid and sustained phosphorylation of STAT3 protein. These data indicate that IL-6 could influence wound healing by inducing keratinocyte migration through the production of a soluble fibroblast-derived factor, and its activity may be associated with STAT3 activation. IL-6-deficient transgenic mice (IL-6 KO) display significantly delayed cutaneous wound healing. To further elucidate the role of IL-6 in skin wound healing, epidermal keratinocyte and dermal fibroblast cells were isolated from neonatal IL-6 KO mice and treated with rmIL-6. It was found that rmIL-6 alone did not significantly modulate the proliferation or migration of cultured IL-6 KO keratinocytes. rmIL-6, however, significantly induced the migration of IL-6 KO keratinocytes (up to 5-fold) when co-cultured with dermal fibroblasts. Culture supernatants from IL-6-treated fibroblasts were also found to induce the migration of keratinocytes to a similar degree. Genomics analysis of treated fibroblasts indicated that rmIL-6 does not induce any known soluble keratinocyte migratory factors. rmIL-6 treatment of fibroblast, however, induced a rapid and sustained phosphorylation of STAT3 protein. These data indicate that IL-6 could influence wound healing by inducing keratinocyte migration through the production of a soluble fibroblast-derived factor, and its activity may be associated with STAT3 activation. IL-6 (10ng per mL)-treated fibroblast-conditioned medium complete keratinocyte growth medium serum-freegrowth factor freekeratinocyte growth medium In the United States, over 6 million individuals develop chronic skin ulcers annually (Singer and Clark, 1999Singer A.J. Clark R.A. Cutaneous wound healing.N Engl J Med. 1999; 341: 738-746Crossref PubMed Scopus (4308) Google Scholar), and the restoration or augmentation of cutaneous wound healing has long been an elusive goal for health care professionals. Based on gross morphology, the skin can be divided into two major components: the epidermis and dermis. The epidermis is primarily made up of keratinocytes, which form stratified layers of increasingly differentiated cells, terminating in the outermost tough, keratin layer known as the stratum corneum. Beneath the epidermis, the dermis is predominantly composed of an elastic collagen matrix, which contains numerous cell types including fibroblasts, and immune cells such as Langerhans cells. Following trauma to the skin, these diverse cell types must interact and cooperate in an orderly sequence of events that, when simplified, includes coagulation, inflammation, formation of a provisional collagen matrix (granulation tissue), re-epithelialization, and remodeling (for a review, seeBello and Phillips, 2000Bello Y.M. Phillips T.J. Recent advances in wound healing.Jama. 2000; 283: 716-718Crossref PubMed Scopus (102) Google Scholar). While much research has been accomplished to describe the many aspects of dermal and epidermal cell activity during wound healing, the precise mechanisms of the interplay between dermal and epidermal tissue during this complex process are not entirely known. IL-6 is a pleiotropic cytokine that is involved in the growth and differentiation of numerous cell types, including those of dermal and epidermal origin (Sehgal, 1990Sehgal P.B. Interleukin-6: Molecular pathophysiology.J Invest Dermatol. 1990; 94: 2S-6SAbstract Full Text PDF PubMed Google Scholar). In the skin it is produced primarily by epidermal keratinocytes, while macrophages, Langerhans cells, and fibroblasts in the dermis represent other sources of the cytokine (Paquet and Pierard, 1996Paquet P. Pierard G.E. Interleukin-6 and the skin.Int Arch Allergy Immunol. 1996; 109: 308-317Crossref PubMed Scopus (103) Google Scholar). Increased levels of IL-6 have been associated with a number of skin pathologies, such as psoriasis (Grossman et al., 1989Grossman R.M. Krueger J. Yourish D. et al.Interleukin 6 is expressed in high levels in psoriatic skin and stimulates proliferation of cultured human keratinocytes.Proc Natl Acad Sci USA. 1989; 86: 6367-6371Crossref PubMed Scopus (685) Google Scholar), scleroderma (Koch et al., 1993Koch A.E. Kronfeld-Harrington L.B. Szekanecz Z. et al.In situ expression of cytokines and cellular adhesion molecules in the skin of patients with systemic sclerosis. Their role in early and late disease.Pathobiology. 1993; 61: 239-246Crossref PubMed Scopus (152) Google Scholar), and systemic lupus erythematosus (Fugger et al., 1989Fugger L. Morling N. Bendtzen K. et al.IL-6 gene polymorphism in rheumatoid arthritis, pauciarticular juvenile rheumatoid arthritis, systemic lupus erythematosus, and in healthy Danes.J Immunogenet. 1989; 16: 461-465Crossref PubMed Scopus (32) Google Scholar). Overexpression of IL-6 in the skin of normal rats induces epidermal proliferation and inflammation (Sawamura et al., 1998Sawamura D. Meng X. Ina S. Sato M. Tamai K. Hanada K. Hashimoto I. Induction of keratinocyte proliferation and lymphocytic infiltration by in vivo introduction of the IL-6 gene into keratinocytes and possibility of keratinocyte gene therapy for inflammatory skin diseases using IL-6 mutant genes.J Immunol. 1998; 161: 5633-5639PubMed Google Scholar), while transgenic mice overexpressing IL-6 display little more than a thickened stratum corneum (Turksen et al., 1992Turksen K. Kupper T. Degenstein L. Williams I. Fuchs E. Interleukin 6: Insights to its function in skin by overexpression in transgenic mice.Proc Natl Acad Sci USA. 1992; 89: 5068-5072Crossref PubMed Scopus (185) Google Scholar). While it seems that the role of IL-6 is well established in disease conditions, relatively little is known about its role in skin repair. It has been well documented that an inflammatory response following cutaneous wounding is a pre-requisite for healing, and inflammatory cytokines, such as IL-6, might be intimately involved in this process. For instance, glucocorticoids decrease the expression of proinflammatory cytokines in wounds, and interfere with wound repair (Hubner and Werner, 1996Hubner G. Werner S. Serum growth factors and proinflammatory cytokines are potent inducers of activin expression in cultured fibroblasts and keratinocytes.Exp Cell Res. 1996; 228: 106-113Crossref PubMed Scopus (69) Google Scholar). Furthermore, elimination of macrophages, a major source of inflammatory cytokines, from a wounded site delays wound healing (Leibovich and Ross, 1975Leibovich S.J. Ross R. The role of the macrophage in wound repair. A study with hydrocortisone and antimacrophage serum.Am J Pathol. 1975; 78: 71-100PubMed Google Scholar). IL-6 is readily detected in cutaneous wounds (Hubner and Werner, 1996Hubner G. Werner S. Serum growth factors and proinflammatory cytokines are potent inducers of activin expression in cultured fibroblasts and keratinocytes.Exp Cell Res. 1996; 228: 106-113Crossref PubMed Scopus (69) Google Scholar;Kondo and Ohshima, 1996Kondo T. Ohshima T. The dynamics of inflammatory cytokines in the healing process of mouse skin wound: A preliminary study for possible wound age determination.Int J Legal Med. 1996; 108: 231-236Crossref PubMed Scopus (142) Google Scholar), and in the supernatant from keratinocyte cultures subjected to in vitro wounds (Sugawara et al., 2001Sugawara T. Gallucci R.M. Simeonova P.P. Luster M.I. Regulation and role of interleukin 6 in wounded human epithelial keratinocytes.Cytokine. 2001; 15: 328-336Crossref PubMed Scopus (74) Google Scholar). Streptozotocin-induced diabetic mice displayed delayed wound healing, and low levels of IL-6 in wound fluid (Fahey et al., 1991Fahey T.J.D. Sadaty A. Jones W.G.D. Barber A. Smoller B. Shires G.T. Diabetes impairs the late inflammatory response to wound healing.J Surg Res. 1991; 50: 308-313Abstract Full Text PDF PubMed Scopus (209) Google Scholar). IL-6 expression in dermal fibroblasts also decreases with age, which may contribute to delayed cutaneous wound healing in the elderly (Goodman and Stein, 1994Goodman L. Stein G.H. Basal and induced amounts of interleukin-6 mRNA decline progressively with age in human fibroblasts.J Biol Chem. 1994; 269: 19250-19255PubMed Google Scholar). Most significantly, IL-6-deficient transgenic mice (IL-6 KO) display significantly delayed cutaneous wound healing compared to wild-type control animals (Gallucci et al., 2001bGallucci R.M. Sugawara T. Yucesoy B. Berryann K. Simeonova P.P. Matheson J.M. Luster M.I. Interleukin-6 treatment augments cutaneous wound healing in immunosuppressed mice.J Interferon Cytokine Res. 2001; 21: 603-609Crossref PubMed Scopus (64) Google Scholar;Lin et al., 2003Lin Z.Q. Kondo T. Ishida Y. Takayasu T. Mukaida N. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice.J Leukoc Biol. 2003; 73: 713-721Crossref PubMed Scopus (354) Google Scholar). Wounds from IL-6-deficient mice displayed multiple defects including delayed re-epithelialization, greatly decreased granulation tissue, inhibited neovasculariziation, and required up to 3-fold longer to heal. In this article, we describe a cooperative interaction between dermal and epidermal cells that sheds light on the role of IL-6 during wound healing. We detail the effects of rmIL-6 on dermal and epidermal cell proliferation and migration in an in vitro culture system utilizing skin cells from neonatal IL-6 KO mice. This model allows for the examination of the effects of this cytokine without the confounding influence of endogenously produced cytokine. We investigate the possible identity of an IL-6-induced fibroblast-derived keratinocyte migratory factor by genomic analysis, and the possible signaling pathways that may lead to its induction. A mechanism by which IL-6 could affect wound healing is through increased mitogenesis of epidermal keratinocytes. To test this hypothesis, freshly isolated epidermal keratinocytes from IL-6 KO mice were incubated with various concentrations (0–500 ng per mL) of rmIL-6 or KGM for 16 h. DNA synthesis was assessed by BrdU uptake using a commercially available ELISA kit. KGM media induced significant DNA synthesis in cultured keratinocytes compared to SFKGM control Figure 1. Treatment with rmIL-6 did not induce a significant increase in BrdU incorporation at any of the concentrations utilized in either SFKGM or KGM. One of the primary activities of wound keratinocytes is migration across the wound bed. IL-6 could affect this process in either a direct manner, or indirectly by inducing the production of migratory factors from nearby cells in the dermis. Notably, the dermis contains numerous fibroblasts, which upon wounding proliferate and are the primary cells found in granulation tissue. To investigate this hypothesis, freshly isolated keratinocytes were seeded into tissue culture-treated transwell inserts (8 μm pore) and exposed to KGM or various concentrations of rmIL-6, with or without co-culture with fibroblasts. The cultures were allowed to incubate for 16 h, and migrating keratinocytes were determined by enumerating the cells that had migrated across the porous membrane via light microscopy. Treatment with rmIL-6 from 1 to 100 ng per mL did not induce migration of keratinocytes across the transwell inserts (data not shown). When keratinocytes from IL-6 KO mice are co-cultured with dermal fibroblasts (also from IL-6 KO mice) and exposed to rmIL-6 however, the keratinocytes readily migrated across transwell inserts in the absence of collagen or other matrix proteins Figure 2. Culture supernatants collected from overnight cultures of fibroblasts treated with rmIL-6 also induce migration of keratinocytes Figure 3. When IL-6-treated fibroblast culture supernatants were heat treated (100°C, 5 min), the motogenic potential of the supernatants was completely abrogated Figure 3.Figure 3Culture supernatants from rmIL-6 fibroblasts induce migration of keratinocytes. Dermal cells from IL-6 KO mice were grown in 75 cm2 tissue culture flasks until approximately 80% confluence, and then treated with the indicated concentrations of rmIL-6 for 16 h in SFKGM at 37°C and 5% CO2. IL-6-treated fibroblast culture supernatants (FCM) were collected and 0.2 μm filtered. FCM was placed in the wells of a 24-well plate, transwell culture inserts (8 μm, tissue culture treated) were placed in wells of the plate, and IL-6 KO keratinocytes were seeded into the transwell enclosures. Plates were allowed to incubate for 16–18 h, after which the transwell membranes were removed, fixed, stained with hematoxylin, and migrating cells were enumerated by light microscopy. Data are expressed as mean±SEM (n=4) number of cells counted in five random fields when viewed at ×20 magnification approximately 800μm (*significantly different from SFKGM control, p<0.05).View Large Image Figure ViewerDownload (PPT) One of the stimuli that induces keratinocyte migration is contact with collagen I from damaged cell matrix in the wound (Pilcher et al., 1997Pilcher B.K. Dumin J.A. Sudbeck B.D. Krane S.M. Welgus H.G. Parks W.C. The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix.J Cell Biol. 1997; 137: 1445-1457Crossref PubMed Scopus (472) Google Scholar). Others have shown that IL-6 alone could induce keratinocyte migration across a collagen matrix (Sano et al., 1999Sano S. Itami S. Takeda K. et al.Keratinocyte-specific ablation of stat3 exhibits impaired skin remodeling, but does not affect skin morphogenesis.EMBO J. 1999; 18: 4657-4668Crossref PubMed Scopus (412) Google Scholar). To determine if FCM could influence epidermal cell migration on a collagen matrix, primary keratinocytes from IL-6 KO mice were prepared as described above, and seeded onto 100 mm collagen I-coated plates. Once confluent, the cells were serum and growth factor starved for 8 h. An in vitro“wound” was produced and the cells were incubated with 10 ng per mL rmIL-6, FCM, or SFKGM for 24 h. Cells migrating across the wound margin were enumerated in specific microscope fields that had been designated and photographed the previous day for comparison. As shown in Figure 4a, b, c, d, rmIL-6 alone induced migration to a minor, but insignificant degree when compared to serum-free media control. FCM, however, induced significant migration across the wound margin Figure 4a, b, c, d, e. One mechanism by which apparent migration could occur is merely by spreading of keratinocytes as a result of proliferation. To test this hypothesis, freshly isolated epidermal keratinocytes from IL-6 KO mice were incubated with SFKGM, FCM with or without 10 ng per mL rmIL-6, or KGM media for 16 h. DNA synthesis was assessed by BrdU uptake using a commercially available ELISA kit as described above. The results of these assays showed that neither non-treated nor IL-6-treated fibroblast culture supernatants induced a significant mitogenic response from keratinocytes as determined by DNA synthesis Figure 5. To determine whether the apparent induction of a fibroblast-derived migratory factor was associated with the proliferation or survival of dermal fibroblasts in response to rmIL-6 treatment, cells were assayed for DNA replication and viability by BrdU uptake (ELISA) and WST (tetrazolium dye) assay respectively. As shown in Figure 6, rmIL-6 did not induce mitogenesis of dermal fibroblasts at concentrations ranging from 1 to 100 ng per mL as determined by BrdU uptake (light bars). WST assay showed a small but significant increase in cell activity at 1.0 ng per mL, but at higher concentrations (i.e., those associated with keratinocyte migration), tetrazolium dye metabolism was not significantly different from control (dark bars). As shown in Figure 3, following heat treatment (100°C for 5 min) IL-6-treated FCM lost all motogenic activity, indicating that the migratory factor may be a protein. In an attempt to identify the mRNA of this IL-6-induced migratory factor, fibroblasts from IL-6 KO mice were incubated for 2 h in the presence of 10 ng per mL rmIL-6. This concentration of cytokine was determined as optimal from transwell migration experiments Figure 2. Total cellular RNA was collected and gene array experiments were conducted (Clontech Atlas Mouse 5k Array, cat #7906-1, Clontech). Two identical genomics experiments were carried out, the array data from identical treatments were averaged utilizing the “average several arrays” function of Atlas Image (Clontech), and then gene expression differences between treatments were determined also utilizing Atlas Image. A gene that varied in expression ≥2-fold was judged to be modulated by treatment. Once candidate genes were identified by this method, the individual candidate gene spots on each array were examined, and RT-PCR analysis was performed for specific genes to eliminate false positives. Analysis of the arrays in this manner did not indicate that rmIL-6 treatment modulated any genes currently identified as soluble keratinocyte migration factors, and these findings were confirmed by RT-PCR analysis (Table I, Table II).Table ICommon soluble keratinocyte migratory factors.FactorReferencesTgf βSantibanez et al., 2000Santibanez J.F. Quintanilla M. Martinez J. Genistein and curcumin block TGF-beta 1-induced u-PA expression and migratory and invasive phenotype in mouse epidermal keratinocytes.Nutr Cancer. 2000; 37: 49-54Crossref PubMed Scopus (33) Google Scholar,Sarret et al., 1992Sarret Y. Woodley D.T. Grigsby K. Wynn K. O'Keefe E.J. 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Stimulation of keratinocyte migration by growth factors.J Dermatol. 1992; 19: 652-653Crossref PubMed Scopus (46) Google Scholar Open table in a new tab Table IIIL-6-induced fibroblast expression of common soluble migratory factor genesIL-6 induced gene expression (% control)FactorArrayaaverage of two arrays, n/a—gene not available on array.RT-PCRbn=3, ±SE.RT-PCR primer sequenceTgfβ100101 (±3.0)5′-tggaccgcaacaacgccatctatgagaaaacc, 5′-tggagctgaagcaatagttggtatccagggctEGF100110 (±4.3)5′-tgttcatcgcctgacaatgg, 5′-gatgatgcttcccgctcagatFgf 710099.0 (±4.0)5′-cataggtgttataatggttttc, 5′-atactgacacggatcctgccaFgf 10100115 (±9.4)5′-tccagtgccgagagtttcagt, 5′-tggctttgacggcaacaacHgf100100 (±1.2)5′-tgcgctcacagtgctgtgaa, 5′-ttcaatttgctagcatctggTgfαn/a102 (±2.3)5′-acctgcaggtttttggtgcag, 5′-ggagggcgctgggcttctcgIgf-1100108 (±3.6)5′-agctggtggatgctcttcagtt, 5′-gcggtgatgtggcattttctGM-CSF100110 (±5.1)5′-tgtggtctacagcctctcagcac, 5′-caaaggggatatcagtcagaaaggtIL-1β10095.1 (±6.1)5′-gaaatgccaccttttgacag, 5′-caaggccacaggtattttgta average of two arrays, n/a—gene not available on array.b n=3, ±SE. Open table in a new tab The signal transduction pathway of the IL-6 receptor (IL-6R) is complex and involves essentially three pathways: JAK/STAT3, ERK1/2, and the more recently described PI3K/Akt pathway. To determine which pathway might be predominantly stimulated by rmIL-6 in IL-6 KO fibroblasts, dermal cells were exposed to the cytokine at various time points, and nuclear protein was isolated from fibroblast cultures. Quantitation of phospho-STAT3, ERK1/2, and Akt proteins was accomplished by western blot. From these analyses, it was found that STAT3 was phosphorylated in a time- and concentration-dependent manner, whereas the phosphorylation state of ERK1/2 and Akt did not appear to be affected by rmIL-6 treatment Figure 7. IL-6-deficient mice display greatly impaired wound healing as evidenced by delayed wound closure, decreased inflammation, granulation tissue formation, neovascularization, and incomplete re-epithelialization (Gallucci et al., 2000Gallucci R.M. Simeonova P.P. Toriumi W. Luster M.I. TNF-alpha regulates transforming growth factor-alpha expression in regenerating murine liver and isolated hepatocytes.J Immunol. 2000; 164: 872-878Crossref PubMed Scopus (63) Google Scholar;Lin et al., 2003Lin Z.Q. Kondo T. Ishida Y. Takayasu T. Mukaida N. Essential involvement of IL-6 in the skin wound-healing process as evidenced by delayed wound healing in IL-6-deficient mice.J Leukoc Biol. 2003; 73: 713-721Crossref PubMed Scopus (354) Google Scholar). This impairment was noted for as long as 15 d, nearly
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