British Journal of Dermatology (1991) 124, 513-518,
ADONIS 000709639100129Y
REVIEW
Epidermal cytokines and their roles in cutaneous wound healing I.A.MCKAY AND I.M.LEIGH Department of Experimental Dermatology, London Hospital Medical College, 56 Ashfield St, London El 2BL, U,K. Accepted for publication 14 January 1991
Summary
Cytokines are small proteins or glycoproteins which are synthesized and secreted by a variety of cell types. Through binding to specific receptors on target cells, these hormone-like products regulate many normal cell activities, including growth and differentiation, migration and immune functions. Within the epidermis, keratinocytes are the major source of cytokines along with melanocytes and Langerhans cells. In response to a variety of injurious stimuli, including ultraviolet irradiation and cutaneous wounding, epidermal keratinocytes may release a number of these regulatory molecules which can then interact directly with receptors on inflammatory cells. Epidermal cytokines can therefore play an important role in the wound-healing process by recruiting polymorphs and monocytes and in encouraging deposition of extracellular matrix proteins by fibroblasts. Keratinocytes can themselves respond to keratinocyte-derived cytokines by dividing and migrating over the wound surface before differentiating into a new stratified epidermis. This review presents the evidence of the production of cytokines by human keratinocytes and their role in the healing of skin wounds.
In normal skin there is a balance between the number of keratinocytes being produced in the basal cell layer and the number of cells being shed at the skin surface, which results in a stable epidermis.' This in turn probably reflects a balance between growth stimulatory and growth inhibitory factors, now known as cytokines. When keratinocytes are grown in vitro they exhibit a hyperproliferative phenotype^ and start to produce many of these cytokines.^ Likewise cytokines can be found in abundance in skin which is inflamed or injured, especially in conditions with epidermal hyperproliferation such as psoriasis.*'*" This has led to the concept of the 'activated keratinocyte':'^ a cell which in culture or in inflammatory skin disease releases a number of cytokines not found in the normal epidermis. Keratinocytes also appear to adopt such an activated phenotype during the process of wound healing. Below we review the cytokines produced by activated keratinocytes before proceeding to a description of their role in wound healing.
factors, interleukins and colony stimulating factors. Most of them were originally identified by their biological activities and were given appropriate functional names which are listed alongside.
Keratinocyte-derived cytokines
Colony stimulating GM-CSF G-CSF M-CSF Interleukin 3
Table 1 lists the cytokines shown to be produced by human keratinocytes, divided into three groups: growth
Table 1. Cytokines produced by human keratinocytes
References Growth factors bFGF PDGF AA and B TGF-a TGF-^ TNF-a Interleukins IL-laand/? IL-6 IL-8
Basic fibroblast growth factor Platelet derived growth factor Transforming growth factor a Transforming growth factor jl Tumour necrosis factor a
8 9 4, 10, 11 11 9
Epidermal cell-derived thymocyte activating factor (ETAF) Epidermal cell natural killer cell augmenting factor (ENKAF) Neutrophil activating protein
12, 13, 14
factors Granulocyte-macrophage CSF Granulocyte CSF Macrophage CSF Multi-CSF
5, 15 16
17 3 18 19
Correspondence: Dr I,A,McKay,
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Stimulatory growth factors Transforming growth factor a (TGF-a) is found in normal epidermis*'"" hut, consistent with the concept of the 'activated keratlnocyte','' elevated levels of TGF-a messenger RNA and protein are produced by keratinocytes in psoriatic epidermis*-^"'^'. This factor is synthesized as a large precursor molecule which is inserted into the keratinocyte membrane and on activation releases a 50amino-acid peptide,'' which will bind to the receptor for epidermal growth factor (EGF), normally found on the surface of basal keratinocytes.^^ Through this mechanism TGF-a induces keratinocyte division and stimulates keratinocyte migration in vitro,'^^ thereby enlarging cell colonies. The fact that TGF-a has heen shown to he a major autocrine growth factor for cultured keratinocytes has led to its testing along with related growth factors in wound healing. Recent evidence suggests that, despite its name. TGF-a does not cause transformation of keratinocytes'^'* hut seems to be the major positive factor involved in normal keratinocyte replication. Basic fibroblast growth factor (bFGF) is produced by keratinocytes in vitro although it is not thought to be secreted by them. However it does stimulate the growth of melanocytes," presumably acting either by direct cellto-cell contact or by the binding of the growth factor to the extracellular matrix in some fashion. Bovine hFGF has been shown to stimulate human keratinocyte growth-^^ and it is likely that human hFGF acts in a similar fashion. Certainly another member of the FGF family, KGF. acts as a mitogen for human keratinocytes. The platelet-derived growth factors (PDGF) differ from bFGF and TGF-a in that keratinocytes produce them but lack receptors for them. PDGF derived from platelets exists as a disulphide-linked heterodimer of A and B chains. Cultured keratinocytes secrete both homodimers (AA and BB)^ although they constitutively express more PDGF-A than PDGF-B. PDGF is a potent chemotactic factor as well as being an important mitogen for fihroblasts and it plays an important role in the development of granulation tissue during the wound-healing process.
very important regulator of inflammation involved both in recruiting macrophages and inhibiting some lymphocyte functions.^' Interleukins Interleukins constitute a class of cytokines originally identified for their role in mediating immunological functions.^" In recent years it has heen shown that keratinocytes produce or respond to a number of interleukins, indicating that keratinocytes themselves are important memhers of the skin immune system. Interleukin 1 is produced by keratinocytes in two forms, IL-la and IL-ljS.^^ Kupper'' has suggested that the IL-1 found in keratinocytes can only he released once the cell is injured. Neither form is usually secreted hut both require activation by serine proteases for their function. Keratinocytes have IL-1 receptors on their surface and it is likely that IL-1 can activate keratinocytes in vivo through an autocrine pathway. Interleukin 6 (IL-6) is synthesized as a 26-kDa peptide which can be secreted from cells in multiple glycosylated forms. In-situ hybridization experiments show the mRNA for IL-6 in the normal epidermis' and, like TGF-a, it is found in elevated quantities in psoriasis and in cultured keratinocytes and skin tumour-cell lines.^'^ Like IL-1, IL-6 is a major mediator of the acute-phase response.^' It is released from injured cells following bacterial and viral infections, UV-irradiation and burns and is found in elevated levels in the sera of patients following surgery.^^'^^ Thus both IL-1 and IL-6 are products of the 'activated keratinocyte'.'' As yet there is little evidence of a role for these factors in wound healing, although it seems likely that they will soon be implicated in the activation of inflammatory cells and in the replication of keratinocytes during this process. Interleukin 8 (IL-8), also known as neutrophil activating protein (NAP),'** belongs to a family that includes /?-thromboglobulin, which mediates both growth stimulatory and inflammatory processes.^'* IL-8 is produced by cultured human dermal fibroblasts and keratinocytes in response to IL-1 or
Inhibitory growth factors Factors produced by keratinocytes which inhibit their own growth include tumour necrosis factor a (TNF-a)^** and transforming growth factor P (TGF-)8).^'' TNF-a shows synergy with another inhibitory factor released by T cells, interferon y (IFN-y).^** in suppressing keratinocyte growth and increasing the expression of intercellular adhesion molecules. TGF-J3, a 25-kDa protein, is a
Colony stimulating factors The colony stimulating factors (CSFs) support the formation of different blood-cell colonies from haemopoietic precursor cells in semi-solid media. Four colony stimulating factors are produced by cultured keratinocytes:^ multi-CSF (IL-3).'' granulocyte-CSF (G-CSF),^ macrophage-CSF (M-CSF)'** and granulocyte/macrophage-CSF
EPIDERMAL CYTOKINFS AND WOUND HFALING
(GM-CSF).'^ The hest characterized in the skin is GMCSF, a 15-kDa protein, which activates mature granulocytes, eosinophils and T cells as well as promoting granulocyte and macrophage differentiation in the bone marrow. There is some evidence that GM-CSF may also play an important role in the maturation of Langerhans cells.
Keratinocytes respond to cytokines produced by other cells Cytokines which have efi'ects on human keratinocytes but are produced by other cell types and not keratinocytes are listed in Table 2. Epidermal growth factor (FGF) was the first cytokine shown to have any effect on the keratinocyte. Although EGF hinds to receptors in human keratinocytes in vitro and promotes both their growth and persistence, it is not found in any significant quantities in normal human epidermis. However, EGF is found in the a granules of platelets and may be released during wound healing.^^ Other cytokines released from inflammatory cells and fibroblasts, blood mononuclear cells and T cells affect keratinocytes in various disease states. These cytokines include KGF^** (keratinocyte growth factor), a memher of the fibroblast growth factor family hut with specificity for epithelial cells, IFN-y^** and IL-4." Interferons a, pV^ and y,^** like TNF-a, reversibly arrest the growth of keratinocytes in culture, whereas KGF is mitogenic.^^ The final effect of cytokines on the growth of keratinocytes clearly depends on a balance between positive and negative factors acting synergistically or antagonistically.
Activated keratinocytes in wound healing
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incisional or excisional wounding activates the injured keratinocytes. Activated keratinocytes may then release variety of factors like IL-1, IL-6 and TGF-a which can bind to receptors on a variety of different inflammatory cells, including the keratinocytes themselves. Activation of those cells in turn may result in the transcription of genes encoding other cytokines such as TNF-a, IL-8 and GM-CSF and their suhsquent release in biologically active forms. Keratinocytes may also become activated by other pathways, possihly dependent on IFN-y release from T cells or TNF-a from monocytes. The nature of the inflammatory infiltrate may determine the degree of keratinocyte activation and resulting level of cytokine induction. Multiple keratinocyte activators, IL-1, IFN-y and TNF-a have been identified and it is possible that one or more of these factors released from damaged keratinocytes is responsible for initiating the process of wound healing.
The role of cytokines in wound healing The healing of skin wounds can be divided into four phases all potentially involving the mediation of cytokines. Inflammation. This involves the release of agents chemotactic for capillary endothelial cells and cells of the immune system, such as lymphocytes, polymorphs and macrophages. Migration. As epidermal epithelial cells (keratinocytes) migrate from hair follicles and from the wound edge towards the centre of the wound, immune cells and capillaries migrate through the underlying dermis into the wound site.
Any damage to keratinocytes by ultraviolet irradiation, thermal burns, bacterial and viral infections or
Deposition. Matrix components such as the collagens, laminin and fibronectin are deposited in the dermis by fibrohlasts and other cell types.
Table 2. Cytokines produced by other cetl types which have effects on human keratinocytes
Maturation. This involves the contraction of the dermis in full-thickness wounds and squamous differentiation of the keratinocytes on the wound surface.
Reference
Factor IL-4 KGF IFN-a IFN-/J] IFN-y EGF TNF-/?
Interleukin 4 Keratinocyte growth factor (FGF 7) Interferon alpha Interferon beta 1 Interferon gamma Epidermal growth factor Lymphotoxin
35 36 37 37 28 23 28
The study of the role of cytokines in wound healing has been subject to the availability of cytokines in pure form. Initially this involved laborious isolation of proteins from cell extracts and supernatants etc. but more recently, pure factors have heen produced by recombinant DNA technology. Early studies focused on the use of EGF"'*" but TGF-a, vaccinia growth factor (VGF),"'
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and PDGF'*^ have been tested more recently. Crude biological extracts such as platelet releasates"** and homogenates or grafts of cultured keratinocytes*' have been used as sources of bioavailable growth factors but their net result depends on the synergism or antagonism of the contained factors. Most of these studies have been performed in animal models as difficulties have been reported with the use of recombinant products on human volunteers. There are problems in the assessment of many of the available studies due to variations in the following factors.
Mimicking of chronic wounds. Many of the clinical problems of wound healing involve chronic non-healing wounds such as leg ulcers and burns but these are very difficult to mimic in animal models. Steroid treated and diabetic (induced) animals have been used but cannot reproduce the underlying vascular problems of a chronic leg ulcer.
enhanced epidermal regeneration and no complications at 1 year following application.** More recently, TGF-a and VGF improved the healing of split thickness incisional wounds and partial thickness burns but this study was limited to two pigs.*' In pig wounds, TGF-J? application caused an increase in connective tissue including elevated collagen levels, a promotion of angiogenesis and an increase in tensile strength,^^ but TGF-fi is pro-inflammatory and causes abnormal epidermal differentiation. It has been suggested that limited effects from the use of individual factors might be overcome by their application in combinations. There have been few studies of this type. Eisinger et al. used extracts of cultured keratinocytes and supernatants from cultured cells on split-thickness wounds in pigs.*^ Five days after application there was more extensive epidermal regeneration in the treated wounds when compared with controls. It seems probable however that this form of treatment has no advantages over the application of cultured skin grafts, which presumably release the same factors but are likely have a superior dressing effect. Lynch et a/.*'' tested the effects of various combinations of purified cytokines on pig wounds. They reported that the combinations of PDGF with either insulin-like growth factor 1 (IGF-1) or TGF-a gave the best results in terms of connective tissue deposition, angiogenesis and collagen content and maturity. Indeed, compared to TGF-^ alone, these combinations of factors also give better epidermal differentiation and reduced inflammation. It seems therefore that combinations of factors will be attractive alternatives to grafting or single factor therapies. Studies on the effects of the interleukins and colony stimulating factors on wound healing are so far lacking. However it seems likely that potent activators of keratinocytes such as IL-1 will have an important role to play in the wound healing process.
Results of wound healing studies to date
The future
Despite slow and intermittent progress, the results of cytokine testing on animal and human wounds parallel the results on keratinocytes in vitro. EGF and TGF-a promote keratinocyte growth and migration both in culture and in wounds.^^•^'•*" The application of a saline solution of mouse EGF to suction blisters did not significantly affect epidermal regeneration^' but healing of split-thickness wounds on pig flank was enhanced by application of EGF in a cream vehicle.*" Simflarly, on human volunteers EGF in Silvadene was found to be superior to vehicle alone in 12 donor sites with
In wound healing studies the challenge of the nineties will be to ascertain which cytokine activities are important. The tools are readily available for such studies: monospecific antisera for immunocytochemistry, cloned DNA probes for in-situ hybridization and biological assays of function. Using this powerful combination it should prove possible to discover which cytokines are released in the initial response to cutaneous wounds: the pattern of cytokines subsequently induced: how much of each cytokine is present and how long they remain active. Only with this information will it be possible to
The animal model Initial experiments were performed on mice or on human volunteers but now the pig has reemerged as the model of choice, both because of the acknowledged similarities between the pig and humans and because the larger wounds can be more easily surveyed during the healing process. The method of applying the cytokines. Cytokines have been applied as wound irrigation in buffered solution and as ointments but the long-term stability and activity of these cytokines has seldom been established. The method of assessing wound healing. The measurement of wound area is complicated by the damage from repeated dressings, difficulties in accurate assessment of the wound edge and distortion from wound contraction.
EPIDERMAL CYTOKINES AND WOUND HEALING
define rational therapeutic approaches to intervention in the would healing process. Meanwhile the cost of recombinant factors excludes their use in routine wound healing management. However, large-scale manufacture of stable active formulations may allow their use in treatment of chronic non-healing wounds or hypertrophic scars.
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Acknowledgments I.McKay gratefully acknowledges support from the Restoration of Appearance and Function Trust (RAFT) and the Emmandjay Trust.
17
References
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1 BuUough WS. The control of mitotic activity in adult mammalian tissues. Biol Rev 1962; 37: 307-42. 2 Leigh [M, Pulford KA. Ramaekers FCS. Lane EB. Psoriasis: maintenance of an intact monolayer basal cell differentiation compartment in spite of hyperproliferation. Br / Derinatol 1985: 113: 53-64. 3 Kupper TS, Horowitz M, Birchall N et al. Hematopoietic. lymphopoietic and proinflammatory cytokines produced by human and murine keratinocytes. In: Endocrine. Metabolic and Immunologic Functions of Keratinocytes (Milstone LM, Edelson RL, eds). Annals NY Acad Sci, Vol. 548. New York Academy of Sciences Publications, 1988: 262-70. 4 Gottlieb AB, Chang CK, Posnett DN et al. Detection of transforming growth factor a in normal, malignant and hyperproliferative human keratinocytes. / Exp Med 1988: 167: 670-5. 5 Grossman RM, Krueger ]. 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: 6367-71. 6 Krueger ]G. Krane JF, Carter M et al. Role of growth factors, cytokines and their receptors in the pathogenesis of psoriasis. / Invest Dermaton 990: 94: 135S-4OS. 7 Kupper TS. The activated keratinocyte: a model for inducible cytokine production by non-bone marrow-derived cells in cutaneous inflammatory and immune responses. / Invest Dermatol 1990: 94: 146S-50S. 8 Halaban R. Langdon R, Birchall N et al. Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. / Cell Biol 1988: 107: 1611-19. 9 AnselJ. Perry P, Brown Jet a/. Cytokine modulation of keratinocyte cytokines. / Invest Dermatol 1990: 94: 101S-7S. 10 Coffey RJ Jr. Derynck R. Wilcox JN et al. Production and autoinduction of transforming growth factor-a in human keratinocytes. Nature 1987: 328: 817-20. 11 Partridge M. Green MR, Langdon JD. Feldmann M. Production of TGF-a and 'IG?-[i by cultured keratinocytes. skin and oral squamous cell carcinomas—potential autocrine regulation of normal and malignant epithelial cell proliferation. Br / Cancer 1989: 60: 542-48. 12 Luger TA. Stadler BM. Luger BM et al. Characteristics of an epidermal cell thymocyte-activating factor (ETAF) produced by human epidermal cells and a human squamous cell carcinoma line. / Invest Dermatol 1983: 8 1 : 187-93. 13 Kupper TS. Ballard DW, Chua AO et al Human keratinocytes
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contain mRNA indistinguishable from monocyte interleukin l a and /? mRNA. / Exp Med 1986: 164: 2095-100. Ristow H-J. A major factor contributing to epidermal proliferation in inflammatory skin diseases appears to be interleukin 1 or a related protein. Proc Natl Acad Sci USA 1987: 84: 1940-4. Kirnbauer R, Kock A. Schwarz T et al. IFN/52, B cell differentiation factor 2 or hybridoma growth factor (IL-6) is expressed and released by human epidermal cells and epidermoid carcinoma cell lines. / Immunol 1989: 142: 1922-8. Larsen CG. Anderson AO, Oppenheim JJ et al. Production of interleukin-8 by human dermal flbroblasts and keratinocytes in response to interleukin-1 or tumour necrosis factor. Immunology 1989: 68: 31-6. Kupper TS. Lee F. Coleman D et al. Keratinocyte derived T-cell growth factor (KTGF) is identical to granulocyte macrophage colony stimulating factor (GM-CSF). / Invest Dermatol 1988: 9 1 : 185-8. Chodakewitz JA, Lacy J, Edwards SE et al. Macrophage colonystimulating factor production by murine and human keratinocytes. / Immunol 1990: 144: 2190-6. Luger TA, Kock A, Kirnbauer R et al Keratinocyte-derived interleukin 3. In: Endocrine. Metabolic and Immunologic Functions of Keratinocytes (Milstone LM, Edelson RL. eds). Annals NY Acad Sci. Vol. 548. New York Academy of Sciences Publications. 1988: 253-61. Elder JT, Fisher GJ, Lindquist PB et al. Overexpression of transforming growth factor ct in psoriatic epidermis. Science 1989: 243: 8 1 1 14. Turbitt ML, Akhurst RJ, White SI. MacKie RM. Localization of elevated transforming growth factor-alpha in psoriatic epidermis. / invest Dermatol 1990: 95: 229-32. King LE Jr. Gates RE, Stoscheck CM, Nanney LB. The EGF/ TGFa receptor in skin. / Invest Dermatvl 1990: 94: 164S-70S. Barrandon Y, Green H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor and epidermal growth factor. Cell 1987: 50: 1131-7. Finzi E, Fleming T. Pierce JH. Retroviral expression of transforming growth factor-alpha does not transform fibroblasts or keratinocytes. / Invest Dermatol 1990: 95: 382-7. O'Keefe EJ. Chiu ML, Payne RE. Stimulation of growth of keratinocytes by bFGF. / Invest Dermatol 1988: 90: 767-9. Pillai S. Bikle DD. Eessalu TE et al Binding and biological effects of tumour necrosis factor alpha on cultured human neonatal foreskin keratinocytes. / Clin Invest 1989: 83: 816-21. Shipley GD, Pittelkow MR. Wille jJ Jr et al. Reversible inhibition of human prokeratinocyte proliferation by type beta transforming growth factor inhibitor in serum free medium. Cancer Res 1986: 46: 2068-71. Symington FW. Lymphotoxin. tumor necrosis factor and gamma interferon are cytostatic for normal human keratinocytes. / Invest Dermatol 1989: 92: 798-805. Quaglino D Jr, Nanney LB, Kennedy R. Davidson JM. Transforming growth factor-^ stimulates wound healing and modulates extracellular matrix gene expression in pig skin. Lab Invest 1990: 63: 307-19. O'Garra A, Umland S. De France T. Christiansen J. B-cell factors' are pleiotropic. Immunol Today 1988: 9: 45-54. Heinrich PC, Castell JV, Andus T. Interleukin-6 and the acute phase response. Biochem ] 1990: 265: 621-36. Nijsten MWN. De Groot ER, Ten Duis HJ. Serum levels of IL-6 and acute phase responses. Lancet 1987: ii: 921. Nishimoto N, Yoshizaki K, Tagoh H et al. Elevation of serum interleukin 6 prior to acute phase proteins on the intlammation by
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surgieal operation. Cliii Immunol Immunopathol 1989: 50: 399401. Stoeckle MY. Barker KA. Two burgeoning families of platelet factor 4-reIated proteins: mediators of the inflammatory response. New Biologist 1990: 2: 313-23. Birchall N. Park L. Kupper T. IL-4 binds to specific cell surface receptors on human keratinocytes and induces colony stimulating factor release and HLA-DR expression. Clin Res 1988: 36: 374A. Finch PW. Rubin ]S. Miki T et al. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 1989:245: 752-5. Yaar M. Karasslk RL. Schnipper LE. Gilchrest BA. Effect of alpha and beta interferon on cultured human keratinocytes. / Invest Dermato] 1985: 85: 70-4. Ben-Ezra I. Sheibani K, Hwang DL. Lev-Ran A. Megakaryocyte synthesis is the source of epidermal growth factor in human platelets. Am ] Pathol 1990: 137: 755-9. Greaves MW. Lack of effect of topically applied epidermal growth factor (EGF) on epidermal growth in man in vivo. Clin Exp Dermatol 1980: 5: 101-3.
40 Brown GL. Curtsinger L. Brightwell IR et al. Enhancement of epidermal regeneration by biosynthetic epidermal growth factor. / Exp Med 1986: 163: 1319-24. 41 Schultz GS. White M. Mitchell R et al. Epithelial wound healing enhanced by transforming growth factor-a and vaccinia growth factor. Science 1987: 235: 350-2. 42 Schweigerer L. Basic fibroblast growth factor as a wound healing hormone. TIPS 1988: 9: 427-8. 43 Gills IP. Melntyre LG. Growth factors and their promising future. / Am Optom Assoc 1989: 60: 442-5. 44 Van Brunt ]. Lessons from wound healing clinical trials. Biotechnology 1989: 7: 15-16. 45 Eisinger M. Sadam S. Silver IA. Flick RB. Growth regulation of skin cells by epidermal cell-derived factors: Implications for wound healing. Proc Natl Acad Sci USA 1988: 85: 1937-41. 46 Lynch SE. Colvin RB. Antoniades HN. Growth factors in wound healing. / Clin Invest 1989: 84: 640-6.
ADONIS 000709639100129Y
REVIEW
Epidermal cytokines and their roles in cutaneous wound healing I.A.MCKAY AND I.M.LEIGH Department of Experimental Dermatology, London Hospital Medical College, 56 Ashfield St, London El 2BL, U,K. Accepted for publication 14 January 1991
Summary
Cytokines are small proteins or glycoproteins which are synthesized and secreted by a variety of cell types. Through binding to specific receptors on target cells, these hormone-like products regulate many normal cell activities, including growth and differentiation, migration and immune functions. Within the epidermis, keratinocytes are the major source of cytokines along with melanocytes and Langerhans cells. In response to a variety of injurious stimuli, including ultraviolet irradiation and cutaneous wounding, epidermal keratinocytes may release a number of these regulatory molecules which can then interact directly with receptors on inflammatory cells. Epidermal cytokines can therefore play an important role in the wound-healing process by recruiting polymorphs and monocytes and in encouraging deposition of extracellular matrix proteins by fibroblasts. Keratinocytes can themselves respond to keratinocyte-derived cytokines by dividing and migrating over the wound surface before differentiating into a new stratified epidermis. This review presents the evidence of the production of cytokines by human keratinocytes and their role in the healing of skin wounds.
In normal skin there is a balance between the number of keratinocytes being produced in the basal cell layer and the number of cells being shed at the skin surface, which results in a stable epidermis.' This in turn probably reflects a balance between growth stimulatory and growth inhibitory factors, now known as cytokines. When keratinocytes are grown in vitro they exhibit a hyperproliferative phenotype^ and start to produce many of these cytokines.^ Likewise cytokines can be found in abundance in skin which is inflamed or injured, especially in conditions with epidermal hyperproliferation such as psoriasis.*'*" This has led to the concept of the 'activated keratinocyte':'^ a cell which in culture or in inflammatory skin disease releases a number of cytokines not found in the normal epidermis. Keratinocytes also appear to adopt such an activated phenotype during the process of wound healing. Below we review the cytokines produced by activated keratinocytes before proceeding to a description of their role in wound healing.
factors, interleukins and colony stimulating factors. Most of them were originally identified by their biological activities and were given appropriate functional names which are listed alongside.
Keratinocyte-derived cytokines
Colony stimulating GM-CSF G-CSF M-CSF Interleukin 3
Table 1 lists the cytokines shown to be produced by human keratinocytes, divided into three groups: growth
Table 1. Cytokines produced by human keratinocytes
References Growth factors bFGF PDGF AA and B TGF-a TGF-^ TNF-a Interleukins IL-laand/? IL-6 IL-8
Basic fibroblast growth factor Platelet derived growth factor Transforming growth factor a Transforming growth factor jl Tumour necrosis factor a
8 9 4, 10, 11 11 9
Epidermal cell-derived thymocyte activating factor (ETAF) Epidermal cell natural killer cell augmenting factor (ENKAF) Neutrophil activating protein
12, 13, 14
factors Granulocyte-macrophage CSF Granulocyte CSF Macrophage CSF Multi-CSF
5, 15 16
17 3 18 19
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Stimulatory growth factors Transforming growth factor a (TGF-a) is found in normal epidermis*'"" hut, consistent with the concept of the 'activated keratlnocyte','' elevated levels of TGF-a messenger RNA and protein are produced by keratinocytes in psoriatic epidermis*-^"'^'. This factor is synthesized as a large precursor molecule which is inserted into the keratinocyte membrane and on activation releases a 50amino-acid peptide,'' which will bind to the receptor for epidermal growth factor (EGF), normally found on the surface of basal keratinocytes.^^ Through this mechanism TGF-a induces keratinocyte division and stimulates keratinocyte migration in vitro,'^^ thereby enlarging cell colonies. The fact that TGF-a has heen shown to he a major autocrine growth factor for cultured keratinocytes has led to its testing along with related growth factors in wound healing. Recent evidence suggests that, despite its name. TGF-a does not cause transformation of keratinocytes'^'* hut seems to be the major positive factor involved in normal keratinocyte replication. Basic fibroblast growth factor (bFGF) is produced by keratinocytes in vitro although it is not thought to be secreted by them. However it does stimulate the growth of melanocytes," presumably acting either by direct cellto-cell contact or by the binding of the growth factor to the extracellular matrix in some fashion. Bovine hFGF has been shown to stimulate human keratinocyte growth-^^ and it is likely that human hFGF acts in a similar fashion. Certainly another member of the FGF family, KGF. acts as a mitogen for human keratinocytes. The platelet-derived growth factors (PDGF) differ from bFGF and TGF-a in that keratinocytes produce them but lack receptors for them. PDGF derived from platelets exists as a disulphide-linked heterodimer of A and B chains. Cultured keratinocytes secrete both homodimers (AA and BB)^ although they constitutively express more PDGF-A than PDGF-B. PDGF is a potent chemotactic factor as well as being an important mitogen for fihroblasts and it plays an important role in the development of granulation tissue during the wound-healing process.
very important regulator of inflammation involved both in recruiting macrophages and inhibiting some lymphocyte functions.^' Interleukins Interleukins constitute a class of cytokines originally identified for their role in mediating immunological functions.^" In recent years it has heen shown that keratinocytes produce or respond to a number of interleukins, indicating that keratinocytes themselves are important memhers of the skin immune system. Interleukin 1 is produced by keratinocytes in two forms, IL-la and IL-ljS.^^ Kupper'' has suggested that the IL-1 found in keratinocytes can only he released once the cell is injured. Neither form is usually secreted hut both require activation by serine proteases for their function. Keratinocytes have IL-1 receptors on their surface and it is likely that IL-1 can activate keratinocytes in vivo through an autocrine pathway. Interleukin 6 (IL-6) is synthesized as a 26-kDa peptide which can be secreted from cells in multiple glycosylated forms. In-situ hybridization experiments show the mRNA for IL-6 in the normal epidermis' and, like TGF-a, it is found in elevated quantities in psoriasis and in cultured keratinocytes and skin tumour-cell lines.^'^ Like IL-1, IL-6 is a major mediator of the acute-phase response.^' It is released from injured cells following bacterial and viral infections, UV-irradiation and burns and is found in elevated levels in the sera of patients following surgery.^^'^^ Thus both IL-1 and IL-6 are products of the 'activated keratinocyte'.'' As yet there is little evidence of a role for these factors in wound healing, although it seems likely that they will soon be implicated in the activation of inflammatory cells and in the replication of keratinocytes during this process. Interleukin 8 (IL-8), also known as neutrophil activating protein (NAP),'** belongs to a family that includes /?-thromboglobulin, which mediates both growth stimulatory and inflammatory processes.^'* IL-8 is produced by cultured human dermal fibroblasts and keratinocytes in response to IL-1 or
Inhibitory growth factors Factors produced by keratinocytes which inhibit their own growth include tumour necrosis factor a (TNF-a)^** and transforming growth factor P (TGF-)8).^'' TNF-a shows synergy with another inhibitory factor released by T cells, interferon y (IFN-y).^** in suppressing keratinocyte growth and increasing the expression of intercellular adhesion molecules. TGF-J3, a 25-kDa protein, is a
Colony stimulating factors The colony stimulating factors (CSFs) support the formation of different blood-cell colonies from haemopoietic precursor cells in semi-solid media. Four colony stimulating factors are produced by cultured keratinocytes:^ multi-CSF (IL-3).'' granulocyte-CSF (G-CSF),^ macrophage-CSF (M-CSF)'** and granulocyte/macrophage-CSF
EPIDERMAL CYTOKINFS AND WOUND HFALING
(GM-CSF).'^ The hest characterized in the skin is GMCSF, a 15-kDa protein, which activates mature granulocytes, eosinophils and T cells as well as promoting granulocyte and macrophage differentiation in the bone marrow. There is some evidence that GM-CSF may also play an important role in the maturation of Langerhans cells.
Keratinocytes respond to cytokines produced by other cells Cytokines which have efi'ects on human keratinocytes but are produced by other cell types and not keratinocytes are listed in Table 2. Epidermal growth factor (FGF) was the first cytokine shown to have any effect on the keratinocyte. Although EGF hinds to receptors in human keratinocytes in vitro and promotes both their growth and persistence, it is not found in any significant quantities in normal human epidermis. However, EGF is found in the a granules of platelets and may be released during wound healing.^^ Other cytokines released from inflammatory cells and fibroblasts, blood mononuclear cells and T cells affect keratinocytes in various disease states. These cytokines include KGF^** (keratinocyte growth factor), a memher of the fibroblast growth factor family hut with specificity for epithelial cells, IFN-y^** and IL-4." Interferons a, pV^ and y,^** like TNF-a, reversibly arrest the growth of keratinocytes in culture, whereas KGF is mitogenic.^^ The final effect of cytokines on the growth of keratinocytes clearly depends on a balance between positive and negative factors acting synergistically or antagonistically.
Activated keratinocytes in wound healing
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incisional or excisional wounding activates the injured keratinocytes. Activated keratinocytes may then release variety of factors like IL-1, IL-6 and TGF-a which can bind to receptors on a variety of different inflammatory cells, including the keratinocytes themselves. Activation of those cells in turn may result in the transcription of genes encoding other cytokines such as TNF-a, IL-8 and GM-CSF and their suhsquent release in biologically active forms. Keratinocytes may also become activated by other pathways, possihly dependent on IFN-y release from T cells or TNF-a from monocytes. The nature of the inflammatory infiltrate may determine the degree of keratinocyte activation and resulting level of cytokine induction. Multiple keratinocyte activators, IL-1, IFN-y and TNF-a have been identified and it is possible that one or more of these factors released from damaged keratinocytes is responsible for initiating the process of wound healing.
The role of cytokines in wound healing The healing of skin wounds can be divided into four phases all potentially involving the mediation of cytokines. Inflammation. This involves the release of agents chemotactic for capillary endothelial cells and cells of the immune system, such as lymphocytes, polymorphs and macrophages. Migration. As epidermal epithelial cells (keratinocytes) migrate from hair follicles and from the wound edge towards the centre of the wound, immune cells and capillaries migrate through the underlying dermis into the wound site.
Any damage to keratinocytes by ultraviolet irradiation, thermal burns, bacterial and viral infections or
Deposition. Matrix components such as the collagens, laminin and fibronectin are deposited in the dermis by fibrohlasts and other cell types.
Table 2. Cytokines produced by other cetl types which have effects on human keratinocytes
Maturation. This involves the contraction of the dermis in full-thickness wounds and squamous differentiation of the keratinocytes on the wound surface.
Reference
Factor IL-4 KGF IFN-a IFN-/J] IFN-y EGF TNF-/?
Interleukin 4 Keratinocyte growth factor (FGF 7) Interferon alpha Interferon beta 1 Interferon gamma Epidermal growth factor Lymphotoxin
35 36 37 37 28 23 28
The study of the role of cytokines in wound healing has been subject to the availability of cytokines in pure form. Initially this involved laborious isolation of proteins from cell extracts and supernatants etc. but more recently, pure factors have heen produced by recombinant DNA technology. Early studies focused on the use of EGF"'*" but TGF-a, vaccinia growth factor (VGF),"'
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and PDGF'*^ have been tested more recently. Crude biological extracts such as platelet releasates"** and homogenates or grafts of cultured keratinocytes*' have been used as sources of bioavailable growth factors but their net result depends on the synergism or antagonism of the contained factors. Most of these studies have been performed in animal models as difficulties have been reported with the use of recombinant products on human volunteers. There are problems in the assessment of many of the available studies due to variations in the following factors.
Mimicking of chronic wounds. Many of the clinical problems of wound healing involve chronic non-healing wounds such as leg ulcers and burns but these are very difficult to mimic in animal models. Steroid treated and diabetic (induced) animals have been used but cannot reproduce the underlying vascular problems of a chronic leg ulcer.
enhanced epidermal regeneration and no complications at 1 year following application.** More recently, TGF-a and VGF improved the healing of split thickness incisional wounds and partial thickness burns but this study was limited to two pigs.*' In pig wounds, TGF-J? application caused an increase in connective tissue including elevated collagen levels, a promotion of angiogenesis and an increase in tensile strength,^^ but TGF-fi is pro-inflammatory and causes abnormal epidermal differentiation. It has been suggested that limited effects from the use of individual factors might be overcome by their application in combinations. There have been few studies of this type. Eisinger et al. used extracts of cultured keratinocytes and supernatants from cultured cells on split-thickness wounds in pigs.*^ Five days after application there was more extensive epidermal regeneration in the treated wounds when compared with controls. It seems probable however that this form of treatment has no advantages over the application of cultured skin grafts, which presumably release the same factors but are likely have a superior dressing effect. Lynch et a/.*'' tested the effects of various combinations of purified cytokines on pig wounds. They reported that the combinations of PDGF with either insulin-like growth factor 1 (IGF-1) or TGF-a gave the best results in terms of connective tissue deposition, angiogenesis and collagen content and maturity. Indeed, compared to TGF-^ alone, these combinations of factors also give better epidermal differentiation and reduced inflammation. It seems therefore that combinations of factors will be attractive alternatives to grafting or single factor therapies. Studies on the effects of the interleukins and colony stimulating factors on wound healing are so far lacking. However it seems likely that potent activators of keratinocytes such as IL-1 will have an important role to play in the wound healing process.
Results of wound healing studies to date
The future
Despite slow and intermittent progress, the results of cytokine testing on animal and human wounds parallel the results on keratinocytes in vitro. EGF and TGF-a promote keratinocyte growth and migration both in culture and in wounds.^^•^'•*" The application of a saline solution of mouse EGF to suction blisters did not significantly affect epidermal regeneration^' but healing of split-thickness wounds on pig flank was enhanced by application of EGF in a cream vehicle.*" Simflarly, on human volunteers EGF in Silvadene was found to be superior to vehicle alone in 12 donor sites with
In wound healing studies the challenge of the nineties will be to ascertain which cytokine activities are important. The tools are readily available for such studies: monospecific antisera for immunocytochemistry, cloned DNA probes for in-situ hybridization and biological assays of function. Using this powerful combination it should prove possible to discover which cytokines are released in the initial response to cutaneous wounds: the pattern of cytokines subsequently induced: how much of each cytokine is present and how long they remain active. Only with this information will it be possible to
The animal model Initial experiments were performed on mice or on human volunteers but now the pig has reemerged as the model of choice, both because of the acknowledged similarities between the pig and humans and because the larger wounds can be more easily surveyed during the healing process. The method of applying the cytokines. Cytokines have been applied as wound irrigation in buffered solution and as ointments but the long-term stability and activity of these cytokines has seldom been established. The method of assessing wound healing. The measurement of wound area is complicated by the damage from repeated dressings, difficulties in accurate assessment of the wound edge and distortion from wound contraction.
EPIDERMAL CYTOKINES AND WOUND HEALING
define rational therapeutic approaches to intervention in the would healing process. Meanwhile the cost of recombinant factors excludes their use in routine wound healing management. However, large-scale manufacture of stable active formulations may allow their use in treatment of chronic non-healing wounds or hypertrophic scars.
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Acknowledgments I.McKay gratefully acknowledges support from the Restoration of Appearance and Function Trust (RAFT) and the Emmandjay Trust.
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References
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