FROM LAB TO PRACTICE
Scar Wars Strategies Target Collagen SAVINA Q. LOW, MD RONALD L. MOY, MD
K
eloids and hypertrophic scars frequently pose chronic and challenging problems. To treat them optimally, one should have insight into the pathogenesis of scar formation and keep abreast of new surgical, pharmacological, and therapeutic approaches to promote desirable outcomes. Current research in this arena is progressive. Novel approaches are being developed to add to our armamentarium of anti-scar weapons. Collagen has been the target of many anti-scarring research efforts. Elaborated by activated fibroblasts, it is the predominant component of scars. Collagen is a molecule composed of three polypeptide chains arranged as a right handed helix. Cross-links between staggered collagen molecules provide stability and strength to the extracellular matrix. Collagen turnover is normally regulated through maintaining a delicate balance between collagen synthesis(basically, transcription of the collagen genes or deoxyribonucleic acid [DNA] sequences to produce procollagen messenger ribonucleic acid [mRNA] sequences followed by translation of mRNA sequences by ribosomes to ultimately produce collagen protein) and collagenase-dependent degradation. During wound healing, the balance is temporarily altered. This can lead to excessive scar formation as seen in keloids and hypertrophic scars.
Collagen Synthesis Inhibitors To win the war against excessive scarring may be to gain control of collagen regulation. Through research in collagen metabolism, various biological response modifiers have been identified that decrease collagen synthesis in dermal fibroblasts.' Several promising examples are amino-acid analogues, procollagen peptides, D-penidlamine, pentoxifylline, corticosteroids,and interferons. In addition, putative cytokine modulators such as soluble cytokine receptors, autoantibodies to cytokines, and cyFrom the Department ofDermatology, University OfCPlifornia-Los Angeles, Los Angeles, California. Address correspondence and reprint requests to: Ronald L Moy, MD, Department of Dermatology, 200 UCLA Medical Center Plaza, Los Angeles, CA 90024-0911.
0 1992 by Elsevier Science Publishing Co., lnc. 0148-0812/92/$5.00
tokine binding molecules may also be useful against excessive scarring. Interference of collagen synthesishas been achieved at the post-translational level using cis-hydroxyproline and azetidine carboxylicacid, structural analogues of proline. They have been reported to inhibit collagen synthesis, cell attachment, and proliferation in cultured human skin fibroblasts derived from healthy controls and patients with active progressive systemic sclerosis.2During translation, the proline analogues are incorporated into newly synthesized polypeptides of procollagen, called pro-a chains. Suffiaent quantities of proline analogues in procollagen can inhibit the formation of stable triple helical procollagen. These destabilizedpro-a chains are secreted from cells into the extracellular matrix at a reduced rate and are more susceptible to degradation by local proteases2As a result, net collagen production is diminished. Systemic administration of cis-hydroxyprolineand azetidine carboxylic acid in animal models of pulmonary fibrosis3and liver cirrhosis4has been reported to decrease excessive collagen deposition. Thus, these proline analogues appear to have clinical potential as anti-scarring agents. Amino-terminal and carboxy-terminal procollagen peptides have also been reported to decrease collagen synthesis in cultured fibroblasts. This was demonstrated in human and bovine skin fibroblastsSand human lung fibr~blasts.~*~ Both procollagen peptides caused sigruficant concentration-dependent decreases in procollagen mRNA and appear to exert their effects pretranslationall^.^,^ Much additional research is required to assess the true clinical potential of these agents. Effective at a later stage of collagen synthesis than proline analogues or procollagen peptides is D-penicillamine. This is the prototypical amino thiol that inhibits the In vitro, D-penicillasynthesis of collagen cro~s-links.~ mine chelates copper and thereby reduces the activity of lysyl oxidase, an enzyme that plays an important role in the formation of cross-links. In vivo, a secondary mechanism of action related to a direct effect on collagen crosslinkingrather than an effect on lysyl oxidase is thought to occur. Because uncross-linked collagen is more susceptible to degradation by collagenases, inhibition of crosslink formation leads to a net decrease in collagen deposi-
981
J Dermatol Surg Oncol
982 LOWANDMOY
1992;18:981- 986
ti0~1.l.~ Systemic D-penicillamine has been used with some success in the treatment of keloids, morphea, and generalized scleroderma,lO*llalthough studies using intralesional administration of D-penicillamine have not been reported. Well-designed clinical trials are required to further assess its clinical applicability. Pentoxifylline, an analogue of methyl xanthine theobromine is another potential weapon against excessive scarring. Pentoxifylline is a hemorrheologic agent used for intermittent claudication that has various effects on erythrocytes as well as dermal fibroblasts.12 It causes dose-dependent inhibition of collagen, fibronectin, and glycosaminoglycan production in human fibroblasts from keloid, scleroderma, morphea,13 and normal skin12 in vitro. Its ability to decrease cellular matrix products provides an impetus for further experimentation in vivo.
Cytokines Cytokines are some of the more novel agents currently under investigation. Two types, interferon (IFN) (-a, -p, and -7) and transforming growth factor-/?(TGF-B), have been clearly shown to have potent but opposite effects on collagen synthesis. Because the former inhibits and the latter stimulates collagen synthesis, promoting or suppressing their activities, respectively, may be useful in controlling collagen synthesis.
Interferons Of the three IFNs (-a, -p, -y), IFN-y appears to be the most well-studied and effective collagen synthesis inhibitor. IFN-y is a lymphokine that exerts a variety of biological effects. It has anti-viral and anti-tumor activity and is a potent immunomodulator. IFN-y binds to its specific IFN-y receptor, a cell membrane-anchored protein, and triggers several chains of events in the cell cytoplasm and the nucleus." Two consequences that are relevant here are inhibition of collagen ~ynthesis'~,'~ and promotion of collagenase production by cultured fibr0b1asts.l~ Initially, IFN-y was demonstrated to inhibit collagen production in human dermal fibroblasts in vitro, and it was proposed to be a potentially effective treatment for diseases characterized by excessive fibrosis.16 Similarly, IFN-y and IFN-/?were also shown to decrease collagen synthesis by dermal fibroblasts in v i t r ~ . ' ~Later, J ~ IFN-y was shown to inhibit fibrosis as a subcutaneous implant in live mice.19 It also effectively inhibited collagen synthesis in scleroderma fibroblasts in vitroZoand improved the symptoms of scleroderma in v i v ~ . ~ l Recently, a double-blind placebo controlled studyU and a non-controlled study" of intralesional IFN-y in the treatment of keloids in human subjects demonstratedsig-
nificant reductions in the size of lesions. In an independent report, reduction in the size and collagen synthesis of a progressively enlarging keloid was reported in one patient who was treated with IFN-a intradermal inject i o n ~This . ~ ~was associated with an in vivo and in vitro decrease in proteoglycan but not fibronectin synthesis by keloidal fibroblasts." These data support the use of IFNs to manage keloids and perhaps other scarring disorders.
Interferon-y Versus Corticosteroids Currently, the most common mode of therapy for hypertrophic scars and keloids is intradermal corticosteroids. The treatment of choice is intralesional triamcinolone acetonide 10 to 20 mg/mL.25 Corticosteroids decrease mRNA for collagen and selectively inhibit collagen synthesis but do not effect noncollagen protein synthesisz6 However, the main disadvantages of using corticosteroids are the possible adverse side effects including atrophy, depigmentation, formation of telangiectasis, and ~lceration.?~ On the other hand, the common adverse effects of keloidal treatment with intralesional IFN-y were reported as headaches and myalgias.U,23In general, the most commonly reported side effects of subcutaneous administration of IFN-y are constitutional. Symptoms include fever, chills, fatigue, myalgias, and headache.28-30Indeed, a well-designed clinical trial comparing the efficacy and associated adverse effects of corticosteroid and interferon intralesional therapy is warranted.
Transforming Growth Factor$ The demonstratedusefulness of IFN-y suggests control of cytokine activity may be a key to successful intervention. In contrast with IFN-y, TGF-pis a cytokine that enhances extracellular matrix production by a variety of cells in vivo31and in v i t r ~ .It~is~associated , ~ ~ with increased procollagen gene expression in keloids,Mand is thought to be important in the pathogenesis of a variety of fibrotic processes such as systemic sclerosis,35eosinophilic fascigeneralized m ~ r p h e a ,hepatic ~~ and glomer~lonephritis.~~ Indeed, TGF-/? is a collagen synthesis stimulator with a myriad of activities related to wound healing. Found mostly in platelets39but also in a variety of cells such as activated macrophages"' and T lymphocytes," TGF-/? stimulates extracellular matrix formation, notably the deposition of fibrone~tin~~ and p r o t e o g l y c a n ~by ~ ~fibroblasts. ,~~ It also inhibits the proteolytic degradation of newly formed matrix proteins by increasing the synthesis of protease inhibitors and decreasing the synthesis and activity of proteases.'2*u
LOW AND MOY 983 COLLAGEN AND EXCESSIVE SCARRING
J Dennatol Surg Oncol 1992;18:981-986
Soluble Cytokine Receptors The inhibition of the effect of TGF-/3on the extracellular matrix may be a viable method to regulate the development of scar tissue. At present, physiologic mechanisms governing cytokine activity are unknown. However, there is evidence that natural soluble cytokine receptors present in vivo" and cytokine receptor-binding antagon i s t ~discovered ~ , ~ ~ in vitro may be important immunoregulators.'s Soluble cytokine receptors for TGF-/349as well as interleukin-2 (IL-2),m-52IL-4,'8s3 IL-6," IFN-y," and tumor necrosis factor (TNF)"*-56 have been described. Some have been characterized to be similar to membrane-bound specific cytokine receptor, but they lack the transmembrane an~hor.'~-~'They can compete with their respective membrane-bound receptor forms for cytokine binding and have been identified in normal human or mouse plasma or urine."*'sss2* A physiological role for these receptors is suggested by reports of elevated IL-2 or TNF soluble receptor levels in systemic lupus erythematosus, atopic eczema, malignancy, and various other disease statesM Most relevant to the present discussion are soluble TGF-Breceptors, which are specificproteoglycanstermed betagly~ans.'~ They are released by a variety of cultured cells including fibroblasts, epithelial cells, myoblasts, adipocytes, and osteosarcoma cells derived from several mammalian species and are found in cell-conditioned culture media, fetal calf serum, and extracellular matriC ~ S They . ~ ~ are suspected to mediate TGF-/3 activity by serving as pericellular reservoirs of bioactive TGF-B." Further research into the putative role of soluble TGF-/3 receptors may yield clinical applications for keloid and hypertrophic scar treatment.
level of TGF-/3 regulation that could be exploited clinically.
Other TGF-&binding Molecules In human serum or plasma, the major binding protein of TGF-/3 is a,-macroglobulin ( C X , - M ) .It~ ~is~present in high concentrations and binds nonspedcally to proteinases as well as various cytokines" such as platelet-derived growth factor.= When bound to certain proteinases such as plasmin, mature a,-M assumes an activated state that has an increased affinity for TGF-/lM When bound to native or activated a,-M, TGF-/3is in a latent form.6z63 Although the physiological function of a,-M is unknown,a,-M may be a cytokine scavenger that mediates rapid plasma clearance of TGF-/3 and directs TGF-/3 to macrophages, fibroblasts, and other cells expressing a,M An immunoregulatory role is further suggested by the association of elevated a,-M concentrations with pregnancy and Additional TGF-P binding molecules that may also modulate this cytokine's activity are decorin, biglycan, and glomerular TGF-/3binding proteins. The two extracellular matrix proteoglycans, decorin and biglycan, inhibit the activity of TGF-/3and may serve as local negative feedback regulators."' Although an inhibitory effect is not known for other TGF-/3 binding molecules such as betaglycans and rat glomerular TGF-/3 binding proteins,71 TGF-Bbinding capacity suggests possible immunomodulatory roles that may be explored.
Summary Autoantibodies to Cytokines Regulation of cytokine activity is thought to be a complex interactive network also involving autoantibodies to cySeveral studies have demonstrated the existence of naturally occurring and disease-associated autoantibodies against several c y t ~ k i n e s . For ~ ~ sexample, ~ plasma levels of autoantibodies to TNF-a are sign&cantly elevated in chronic disease and inflammatory state^.^^,^ In addition, autoantibodies to IFN-y61 and IL158 have been reported in healthy blood donors. Although autoantibodies to TGF-Phave not been described, intravenous administration of antiserum against TGF-/3 has been shown to decrease excessive extracellular matrix production in an animal model of proliferative gl~merulonephritis.~~ Like soluble cytokine receptors, autoantibodies might serve as natural cytokine-specific camers or inhibit0rs.5~These findings suggest another
Hypertrophic scarring and keloid formation are clinical problems with effectively limited solutions. Although numerous methods have been devised to combat them, this articlefocuses on promising pharmacologic strategies that target collagen metabolism. Laboratory investigations of amino-acid analogs, procollagen peptides, Dpenicillamine, and pentoxifylline have demonstrated them to be effective inhibitors of collagen synthesis in cultured cells and/or in animal models. Clinical trials of intralesional administration of interferons have shown impressivereductions in the size and collagen production of keloids. Furthermore, interference of extracellular matrix-enhancing cytokines, such as TGF-/3, may be an effective solution to keloids and hypertrophic scars. Additional research of soluble cytokine receptors, autoantibodies to cytokines, cytokine receptor antagonists, and cytokine-binding molecules may lead to the development of better therapeutic agents.
984 LOWANDMOY
J Dermatol Surg Oncol 1992;18:981-986
References 1. Uitto J, Tan EML, Ryhlnen L. Inhibition of collagen accumulation in fibrotic processes: review of pharmacological agents and new approaches with amino acids and their analogues. J Invest Dermatol 1982;79:113s-120s. 2. Tan EML, Ryhlnen L, Uitto J. Proline analogues inhibit human skin fibroblast growth collagen production in culture. J Invest Dermatol 1983;80:261-7. 3. Riley DJ, Kerr JS, Berg RA, et al. Prevention of bleomycininduced pulmonary fibrosis in the hamster by cis-4-hydroxy-L-proline. Am Rev Respir Dis 1981;123:388-93. 4. Rojkind M. Inhibition of liver fibrosisby L-azetidine-2-carboxylic acid in rats treated with carbon tetrachloride.J Clin Invest 1973;52:2451-6. 5. Wiestner M, Krieg T, Dietrich H, Glanville RW, Fietzek P, Miiller PK. Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures. J Biol Chem 1979;254:7016-23. Wu CH, Donovan CB, Wu GY. Evidence for pretransla6. tional regulation of collagen synthesis by procollagen propeptides. J Biol Chem 1986;261:10482-4. 7. Wu CH, Walton CM, Wu GY. Propeptide-mediated regulation of procollagen synthesis in IMR-90 human lung fibroblast cell cultures. J Biol Chem 1991;266:2983-7. 8. Siegel RC. Collagen cross-linking:effect of D-penicillamine on cross-linking in vitro. J Biol Chem 1977;252:254-9. 9. Vater CA, Harris Jr ED, Siegel RC. Native cross-links in collagen fibrils induce resistance to human synovial collagenase. Biochem J 1979;181:639-45. 10. Asboe-Hansen G. Treatment of generalized scleroderma: updated results. Acta-Dermatovenereol1979;59:465 -467. 11. Moynahan EJ. Penicillamine in the treatment of morphoea and keloid in children. Postgrad Med J (Suppl); 1974;3940. 12. Berman B, Duncan MR. Pentoxifylline inhibits normal human dermal fibroblast in vitro proliferation, collagen, glycosaminoglycan, and fibronectin production, and increases collagenase activity. J Invest Dermatol 1989; 92~605-10. 13. Berman B, Duncan MR. Pentoxifylline inhibits the proliferation of human fibroblasts derived from keloid, scleroderma and morphoea skin and their production of collagen, glycosaminoglycans and fibronectin. Br J Dermatol 1990; 1231339-46. 14. Rubinstein M, Novick D, Fischer DG. The human interferon-y receptor system. Immunol Rev 1987;97:29-50. 15. Jimenez SA, Freundlich B, Rosenbloom J. Selective inhibition of human diploid fibroblast collagen synthesis by interferons. J Clin Invest 1984;74:1112-6. 16. Duncan MR,Berman B. y-Interferon is the lymphokine and /?-interferon the monokine responsible for inhibition of fibroblast collagen production and late but not early fibroblast proliferation. J Exp Med 1985;162:516-27.
17. Duncan MR, Berman B. Differential regulation of glycosaminoglycan, fibronectin, and collagenase production in cultured human dermal fibroblasts by interferon-alpha, -beta, and -gamma. Arch Dermatol Res 1989;281:11-8. 18. Duncan MR, Berman B. Persistence of a reduced-collagenproducing phenotype in cultured scleroderma fibroblasts after short-term exposure to interferons. J Clin Invest 1987;79:1318-24. 19. Granstein RD, Murphy GF, Margolis RJ, Byme MH, Amento EP. Gamma-interferon inhibits collagen synthesis in vivo in the mouse. J Clin Invest 1987;79:1254-8. 20. Kiihiri V-M, Heino J, Vuorio T, Vuorio E. Interferon-a and interferon-y reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture. Biochim Biophys Acta 1988;968:45-50. 21. Kahan A, Amor B, Menkes CJ, Strauch G. Recombinant interferon-y in the treatment of systemic sclerosis. Am J Med 1989;87273-7. 22. Granstein RD, Rook A, Flotte TJ, et al. Controlled trial of intralesional recombinant interferon-y in the treatment of keloidal scarring. Arch Dermatol 1990;126:1295-302. 23. Larrabee WF Jr, East CA, Jaffe HS, Stephenson C, Peterson KE. Intralesional interferon gamma treatment for keloids and hypertrophic scars. Arch Otolaryngol Head Neck Surg 1990;116:1159-62. 24. Berman 8, Duncan MR. Short-term keloid treatment in vivo with human interferon alfa-2b results in a selective and persistent normalization of keloidal fibroblast collagen, glycosaminoglycan, and collagenase production in vitro. J Am Acad Dermatol 1989;21:694-702. 25. Goslen JB. Wound healing after cosmetic surgery. In: Coleman 111 WP, Hanke CW, Alt TH, Asken S, eds. Cosmetic Surgery of the Skin Principles and Techniques. Philadelphia: BC Decker, 1991:47-63. 26. Pinnell SR. Regulation of collagen synthesis. J Invest Dermatol (suppl) 1982;79:73-6. 27. Murray JC, Pollack SV, Pinnell SR. Keloids: a review. J Am Acad Dermatol 1981;4:461-70. 28. Russo D, Fanin R, Zuffa E, et al. Treatment of Ph+ chronic myeloid leukemia by gamma interferon. Blut 1989;59:1520. 29. Maluish AE, Urba WJ, Longo DL, et al. The determination of an immunologically active dose of interferon-gamma in patients with melanoma. J Clin Oncol 1988;6:434-45. 30. Aulitzky WE,Aulitzky W, Gastl G, et al. Acute effects of single doses of recombinant interferon-y on blood cell counts and lymphocyte subsets in patients with advanced renal cell cancer. J Interferon Res 1989;9:425-33. 31. Roberts AB, Spom MB, Assoian RK, et al. Transforming growth factor type 8:rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 1986;83:4167-71. 32. Ignotz RA,MassaguC J. Transforming growth factor-Bstimdates the expression of fibronectin and collagen and their
1Dermatol Surg Oncol 1992;18:981-986
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
incorporation into the extracellular matrix. J Biol Chem 1986;261:4337-45. Ignotz RA, Endo T, MassaguC J. Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor$. J Biol Chem 1987;262:6443-6. PeItonen J, Hsiao LL, Jaakkola S, et al. Activation of collagen gene expression in keloids co-localization of types I and VI collagen and transforminggrowth factor-@ mRNA. J Invest Dermatol 1991;97240-8. Gruschwitz M, Miiller PU, Sepp N, Hofer E, Fontana A, Wick G. Transcription and expression of transforming growth factor type beta in the skin of progmsive systemic sclerosis a mediator of fibrosis? J Invest Dermatol 1990; 94~197-203. Peltonen J, K5hiiri L, Jaakkola S, et al. Evaluation of transforming growth factor B and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization. J Invest Dermatol 1990;94:365-71. Czaja MJ, Weiner FR, Flanders KC, et al. In vitro and in vivo association of transforming growth factor-fl with hepatic fibrosis. J Cell Biol 1989;108:2477-82. Border WA Okuda S, Languino LR, Spom MB,Ruoslahti E. Suppression of experiznental glomdonephritis by antiserum against transforminggrowth factor @. Nature 1990; 346371 -4. Assoian RK, Komoriya A, Meyers CA, Miller DM, Spom MB. Transforming growth factor-/? in human platelets: identification of a major storage site, purification and characterization.J Biol Chem 1983;258:7155-7160. Assoian RK, FleurdelysBE, StevensonHC, et al. Expression and secretion of type b transforming growth factor by activated human macrophages. Proc Natl Acad Sci USA 1987; 84:6020 -4. Kerhl JH,Wakefield LM, Roberts AB, et al. Production of transforminggrowth factors by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986;163:1037-50. Spom MB, Roberts AB. Transforming growth factor-fi multiple actions and potential clinical applications. JAMA 1989;262:938-41. Falanga V, Tiegs SL, Alstadt SP, Roberts AB, Spom MB. Transforming growth factor-beta: selective inaease in glycosaminoglycan synthesis by cultures of fibroblasts from patients with progressive systemic sclerosis. J Invest Dermatol 1987;89: 100-4. Spom MB, Roberts AB, Wakefield LM,de CrombruggheB. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol 1987; 105:1039 -45. Novick D, Engelmann H, Wallach D, Rubinstein M. Soluble cytokine receptorsare present in normal human urine. J Exp Med 1989;170:1409-14. H~~IIUXII CH, W ~ ~ CCJ, O XArend WP, et al. Interleukin-1 receptor antagonist activityof a human interleukin-1inhibitor. Nature 1990;343:336-40.
LOW AND MOY 985 COLLAGEN AND EXCESSIVE SCARRING
47. EisenbergSP, Evans RJ, Arend WP,et al. Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature 1990; 343~341-6. 48. Femandez-BotranR. Soluble cytokine receptors: their role in immunoregulation.FASEB 1991;5:2567-74. 49. Andres JL, Stanley K, Cheifetz S, MassaguC J. Membraneanchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-j?. J Cell Biol1989;109:3 137- 45. 50. Rubin LA, Kurman CC, Fritz ME, et al. Solubleinterleukin 2 receptorsare released from activatedhuman lymphoid cells in vitro. J Immun01 1985;135:3172-7. 51. Treiger BF, Leonard WJ, Svetlik P, Rubin LA, Nelson DL, Greene WC. A secreted form of the human interleukin 2 receptor encoded by an “anchor minus” cDNA. J Immunol 1986;136:4099- 105. 52. Osawa H, Josimovic-Alasevic0, Diamantstein T. Interleukin 2 receptors are released by cells in vim and in vivo. I. Detection of soluble IL 2 receptors in cell culture supematants and in the serum of mice by an immunoradiometric m y . EW J Immunol 1986;16:467-9. 53. MosIey B, BeckmannMP,March CJ, et al. The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms. Cell 1989; 59~335-48. 54. Peetre C, Thysell H, Grubb A, Olsson I. A tumor necrosis factor binding protein is present in human biological fluids. EUI J Haemat01 1988;41:414-9. 55. Engelmann H, Aderka D, Rubinstein M, Rotman D, Wallach D. A tumor necrosis factor-bindingprotein purified to homogeneity from human urine protects cells from tumor necrosis factor toxiaty. J Biol Chem 1989;264:11974-80. 56. Schall TJ, Lewis M, Koller KJ, et al. Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 1990;61:361- 70. 57. Bendtzen K, Svenson M, JBnsson V, Hippe E. Autoantibodies to cytokines-friends or foes? Immunol Today 1990; 11:167-9. 58. Svenson M, Poulsen LK, Fomsgaard A, Bendtzen K. IgG autoantibodies against interleukin la in sera of normal ind i v i d d . Scand J Imm~11011989;29:489-92. 59. Bendtzen K, Svenson M, Fomsgaard A, Poulsen LK. Native inhibitors (autoantibodies) of IL-la and TNF. Immunol Today 1989;10:222. 60. Fomsgaard A, Svenson M, Bendtzen K. Auto-antibodies to lumour nec-rosis factor a in healthy humans and patients with inaammatory diseases and gram-negative bacterial infections. Scand J Immun011989;30:219-3. 61 Ross C, Hansen MB,Schyberg T, Berg K. Autoantibodiesto crude human leucocyte interferon (IFN), native human IFN, recombinant human IFN-alpha 2b and human IFNgamma in healthy blood donors. Clin Exp Immunol. 1990;82:57-62.
986 LOW AND MOY
62. O’Connor-McCourt MD, Wakefield LM. Latent transforming growth factor$ in serum: a specific complex with a,macroglobulin. J Biol Chem 1987;262: 14090- 9. 63. Huang SS, OGrady P, Huang JS. Human transforming growth factor /3 a,-macroglobulin complex is a latent form of transforming growth factor /3. J Biol Chem 1988;263:1535-41. 64. LaMarre J,Wollenberg GK, Gonias SL, Hayes MA. Cytokine binding and clearance properties of proteinase-activated a,-macroglobulins. Lab Invest 1991;65:3- 14. 65. Huang JS,Huang SS, Deuel TF. Specificcovalent binding of platelet-derived growth factor to human plasma az-macroglobulin. Proc Natl Acad Sci USA 1984;81:342-6. 66. LaMarre J,Wollenberg GK, Gonias SL, Hayes MA. Reaction of a,-macroglobulin with plasmin increases binding of transforming growth factors-fl and 82. Biochim Biophys Acta 1990;1091:197-204.
-
Dennatol Surg Oncol 1992;18:981-986 67. LaMarre J,Hayes MA, Wollenberg GK, Hussaini I, Hall SW, Gonias SL. An a,-macroglobulin receptor-dependent mechanism for the plasm clearance of transforming growth factor-/3l in mice. J Clin Invest 1991;8739-44. 68. Stimson WH.Quantitation of a new serum a-macroglobulin in malignant disease, steroid treatment, pregnancy and normal subjects. J Endocr 1974;61:30-31. 69. Stimson WH.Variations in the level of a pregnancy-assoaated a-macroglobulin in patients with cancer. J Clin Path 1975;28:868-71. 70. Yamaguchi Y, Mann DM, Ruoslahti E. Negative regulation of transforming growth factor-/3 by the proteoglycan decorin. Nature 1990;346:281-4. 71. MacKay K, Robbms AR, Bruce MD, Danielpour D. Identification of disulfide-linked transforming growth factor-/3l-speci6c binding proteins in rat glomeruli. J Biol Chem 1990;265:9351-6.
Scar Wars Strategies Target Collagen SAVINA Q. LOW, MD RONALD L. MOY, MD
K
eloids and hypertrophic scars frequently pose chronic and challenging problems. To treat them optimally, one should have insight into the pathogenesis of scar formation and keep abreast of new surgical, pharmacological, and therapeutic approaches to promote desirable outcomes. Current research in this arena is progressive. Novel approaches are being developed to add to our armamentarium of anti-scar weapons. Collagen has been the target of many anti-scarring research efforts. Elaborated by activated fibroblasts, it is the predominant component of scars. Collagen is a molecule composed of three polypeptide chains arranged as a right handed helix. Cross-links between staggered collagen molecules provide stability and strength to the extracellular matrix. Collagen turnover is normally regulated through maintaining a delicate balance between collagen synthesis(basically, transcription of the collagen genes or deoxyribonucleic acid [DNA] sequences to produce procollagen messenger ribonucleic acid [mRNA] sequences followed by translation of mRNA sequences by ribosomes to ultimately produce collagen protein) and collagenase-dependent degradation. During wound healing, the balance is temporarily altered. This can lead to excessive scar formation as seen in keloids and hypertrophic scars.
Collagen Synthesis Inhibitors To win the war against excessive scarring may be to gain control of collagen regulation. Through research in collagen metabolism, various biological response modifiers have been identified that decrease collagen synthesis in dermal fibroblasts.' Several promising examples are amino-acid analogues, procollagen peptides, D-penidlamine, pentoxifylline, corticosteroids,and interferons. In addition, putative cytokine modulators such as soluble cytokine receptors, autoantibodies to cytokines, and cyFrom the Department ofDermatology, University OfCPlifornia-Los Angeles, Los Angeles, California. Address correspondence and reprint requests to: Ronald L Moy, MD, Department of Dermatology, 200 UCLA Medical Center Plaza, Los Angeles, CA 90024-0911.
0 1992 by Elsevier Science Publishing Co., lnc. 0148-0812/92/$5.00
tokine binding molecules may also be useful against excessive scarring. Interference of collagen synthesishas been achieved at the post-translational level using cis-hydroxyproline and azetidine carboxylicacid, structural analogues of proline. They have been reported to inhibit collagen synthesis, cell attachment, and proliferation in cultured human skin fibroblasts derived from healthy controls and patients with active progressive systemic sclerosis.2During translation, the proline analogues are incorporated into newly synthesized polypeptides of procollagen, called pro-a chains. Suffiaent quantities of proline analogues in procollagen can inhibit the formation of stable triple helical procollagen. These destabilizedpro-a chains are secreted from cells into the extracellular matrix at a reduced rate and are more susceptible to degradation by local proteases2As a result, net collagen production is diminished. Systemic administration of cis-hydroxyprolineand azetidine carboxylic acid in animal models of pulmonary fibrosis3and liver cirrhosis4has been reported to decrease excessive collagen deposition. Thus, these proline analogues appear to have clinical potential as anti-scarring agents. Amino-terminal and carboxy-terminal procollagen peptides have also been reported to decrease collagen synthesis in cultured fibroblasts. This was demonstrated in human and bovine skin fibroblastsSand human lung fibr~blasts.~*~ Both procollagen peptides caused sigruficant concentration-dependent decreases in procollagen mRNA and appear to exert their effects pretranslationall^.^,^ Much additional research is required to assess the true clinical potential of these agents. Effective at a later stage of collagen synthesis than proline analogues or procollagen peptides is D-penicillamine. This is the prototypical amino thiol that inhibits the In vitro, D-penicillasynthesis of collagen cro~s-links.~ mine chelates copper and thereby reduces the activity of lysyl oxidase, an enzyme that plays an important role in the formation of cross-links. In vivo, a secondary mechanism of action related to a direct effect on collagen crosslinkingrather than an effect on lysyl oxidase is thought to occur. Because uncross-linked collagen is more susceptible to degradation by collagenases, inhibition of crosslink formation leads to a net decrease in collagen deposi-
981
J Dermatol Surg Oncol
982 LOWANDMOY
1992;18:981- 986
ti0~1.l.~ Systemic D-penicillamine has been used with some success in the treatment of keloids, morphea, and generalized scleroderma,lO*llalthough studies using intralesional administration of D-penicillamine have not been reported. Well-designed clinical trials are required to further assess its clinical applicability. Pentoxifylline, an analogue of methyl xanthine theobromine is another potential weapon against excessive scarring. Pentoxifylline is a hemorrheologic agent used for intermittent claudication that has various effects on erythrocytes as well as dermal fibroblasts.12 It causes dose-dependent inhibition of collagen, fibronectin, and glycosaminoglycan production in human fibroblasts from keloid, scleroderma, morphea,13 and normal skin12 in vitro. Its ability to decrease cellular matrix products provides an impetus for further experimentation in vivo.
Cytokines Cytokines are some of the more novel agents currently under investigation. Two types, interferon (IFN) (-a, -p, and -7) and transforming growth factor-/?(TGF-B), have been clearly shown to have potent but opposite effects on collagen synthesis. Because the former inhibits and the latter stimulates collagen synthesis, promoting or suppressing their activities, respectively, may be useful in controlling collagen synthesis.
Interferons Of the three IFNs (-a, -p, -y), IFN-y appears to be the most well-studied and effective collagen synthesis inhibitor. IFN-y is a lymphokine that exerts a variety of biological effects. It has anti-viral and anti-tumor activity and is a potent immunomodulator. IFN-y binds to its specific IFN-y receptor, a cell membrane-anchored protein, and triggers several chains of events in the cell cytoplasm and the nucleus." Two consequences that are relevant here are inhibition of collagen ~ynthesis'~,'~ and promotion of collagenase production by cultured fibr0b1asts.l~ Initially, IFN-y was demonstrated to inhibit collagen production in human dermal fibroblasts in vitro, and it was proposed to be a potentially effective treatment for diseases characterized by excessive fibrosis.16 Similarly, IFN-y and IFN-/?were also shown to decrease collagen synthesis by dermal fibroblasts in v i t r ~ . ' ~Later, J ~ IFN-y was shown to inhibit fibrosis as a subcutaneous implant in live mice.19 It also effectively inhibited collagen synthesis in scleroderma fibroblasts in vitroZoand improved the symptoms of scleroderma in v i v ~ . ~ l Recently, a double-blind placebo controlled studyU and a non-controlled study" of intralesional IFN-y in the treatment of keloids in human subjects demonstratedsig-
nificant reductions in the size of lesions. In an independent report, reduction in the size and collagen synthesis of a progressively enlarging keloid was reported in one patient who was treated with IFN-a intradermal inject i o n ~This . ~ ~was associated with an in vivo and in vitro decrease in proteoglycan but not fibronectin synthesis by keloidal fibroblasts." These data support the use of IFNs to manage keloids and perhaps other scarring disorders.
Interferon-y Versus Corticosteroids Currently, the most common mode of therapy for hypertrophic scars and keloids is intradermal corticosteroids. The treatment of choice is intralesional triamcinolone acetonide 10 to 20 mg/mL.25 Corticosteroids decrease mRNA for collagen and selectively inhibit collagen synthesis but do not effect noncollagen protein synthesisz6 However, the main disadvantages of using corticosteroids are the possible adverse side effects including atrophy, depigmentation, formation of telangiectasis, and ~lceration.?~ On the other hand, the common adverse effects of keloidal treatment with intralesional IFN-y were reported as headaches and myalgias.U,23In general, the most commonly reported side effects of subcutaneous administration of IFN-y are constitutional. Symptoms include fever, chills, fatigue, myalgias, and headache.28-30Indeed, a well-designed clinical trial comparing the efficacy and associated adverse effects of corticosteroid and interferon intralesional therapy is warranted.
Transforming Growth Factor$ The demonstratedusefulness of IFN-y suggests control of cytokine activity may be a key to successful intervention. In contrast with IFN-y, TGF-pis a cytokine that enhances extracellular matrix production by a variety of cells in vivo31and in v i t r ~ .It~is~associated , ~ ~ with increased procollagen gene expression in keloids,Mand is thought to be important in the pathogenesis of a variety of fibrotic processes such as systemic sclerosis,35eosinophilic fascigeneralized m ~ r p h e a ,hepatic ~~ and glomer~lonephritis.~~ Indeed, TGF-/? is a collagen synthesis stimulator with a myriad of activities related to wound healing. Found mostly in platelets39but also in a variety of cells such as activated macrophages"' and T lymphocytes," TGF-/? stimulates extracellular matrix formation, notably the deposition of fibrone~tin~~ and p r o t e o g l y c a n ~by ~ ~fibroblasts. ,~~ It also inhibits the proteolytic degradation of newly formed matrix proteins by increasing the synthesis of protease inhibitors and decreasing the synthesis and activity of proteases.'2*u
LOW AND MOY 983 COLLAGEN AND EXCESSIVE SCARRING
J Dennatol Surg Oncol 1992;18:981-986
Soluble Cytokine Receptors The inhibition of the effect of TGF-/3on the extracellular matrix may be a viable method to regulate the development of scar tissue. At present, physiologic mechanisms governing cytokine activity are unknown. However, there is evidence that natural soluble cytokine receptors present in vivo" and cytokine receptor-binding antagon i s t ~discovered ~ , ~ ~ in vitro may be important immunoregulators.'s Soluble cytokine receptors for TGF-/349as well as interleukin-2 (IL-2),m-52IL-4,'8s3 IL-6," IFN-y," and tumor necrosis factor (TNF)"*-56 have been described. Some have been characterized to be similar to membrane-bound specific cytokine receptor, but they lack the transmembrane an~hor.'~-~'They can compete with their respective membrane-bound receptor forms for cytokine binding and have been identified in normal human or mouse plasma or urine."*'sss2* A physiological role for these receptors is suggested by reports of elevated IL-2 or TNF soluble receptor levels in systemic lupus erythematosus, atopic eczema, malignancy, and various other disease statesM Most relevant to the present discussion are soluble TGF-Breceptors, which are specificproteoglycanstermed betagly~ans.'~ They are released by a variety of cultured cells including fibroblasts, epithelial cells, myoblasts, adipocytes, and osteosarcoma cells derived from several mammalian species and are found in cell-conditioned culture media, fetal calf serum, and extracellular matriC ~ S They . ~ ~ are suspected to mediate TGF-/3 activity by serving as pericellular reservoirs of bioactive TGF-B." Further research into the putative role of soluble TGF-/3 receptors may yield clinical applications for keloid and hypertrophic scar treatment.
level of TGF-/3 regulation that could be exploited clinically.
Other TGF-&binding Molecules In human serum or plasma, the major binding protein of TGF-/3 is a,-macroglobulin ( C X , - M ) .It~ ~is~present in high concentrations and binds nonspedcally to proteinases as well as various cytokines" such as platelet-derived growth factor.= When bound to certain proteinases such as plasmin, mature a,-M assumes an activated state that has an increased affinity for TGF-/lM When bound to native or activated a,-M, TGF-/3is in a latent form.6z63 Although the physiological function of a,-M is unknown,a,-M may be a cytokine scavenger that mediates rapid plasma clearance of TGF-/3 and directs TGF-/3 to macrophages, fibroblasts, and other cells expressing a,M An immunoregulatory role is further suggested by the association of elevated a,-M concentrations with pregnancy and Additional TGF-P binding molecules that may also modulate this cytokine's activity are decorin, biglycan, and glomerular TGF-/3binding proteins. The two extracellular matrix proteoglycans, decorin and biglycan, inhibit the activity of TGF-/3and may serve as local negative feedback regulators."' Although an inhibitory effect is not known for other TGF-/3 binding molecules such as betaglycans and rat glomerular TGF-/3 binding proteins,71 TGF-Bbinding capacity suggests possible immunomodulatory roles that may be explored.
Summary Autoantibodies to Cytokines Regulation of cytokine activity is thought to be a complex interactive network also involving autoantibodies to cySeveral studies have demonstrated the existence of naturally occurring and disease-associated autoantibodies against several c y t ~ k i n e s . For ~ ~ sexample, ~ plasma levels of autoantibodies to TNF-a are sign&cantly elevated in chronic disease and inflammatory state^.^^,^ In addition, autoantibodies to IFN-y61 and IL158 have been reported in healthy blood donors. Although autoantibodies to TGF-Phave not been described, intravenous administration of antiserum against TGF-/3 has been shown to decrease excessive extracellular matrix production in an animal model of proliferative gl~merulonephritis.~~ Like soluble cytokine receptors, autoantibodies might serve as natural cytokine-specific camers or inhibit0rs.5~These findings suggest another
Hypertrophic scarring and keloid formation are clinical problems with effectively limited solutions. Although numerous methods have been devised to combat them, this articlefocuses on promising pharmacologic strategies that target collagen metabolism. Laboratory investigations of amino-acid analogs, procollagen peptides, Dpenicillamine, and pentoxifylline have demonstrated them to be effective inhibitors of collagen synthesis in cultured cells and/or in animal models. Clinical trials of intralesional administration of interferons have shown impressivereductions in the size and collagen production of keloids. Furthermore, interference of extracellular matrix-enhancing cytokines, such as TGF-/3, may be an effective solution to keloids and hypertrophic scars. Additional research of soluble cytokine receptors, autoantibodies to cytokines, cytokine receptor antagonists, and cytokine-binding molecules may lead to the development of better therapeutic agents.
984 LOWANDMOY
J Dermatol Surg Oncol 1992;18:981-986
References 1. Uitto J, Tan EML, Ryhlnen L. Inhibition of collagen accumulation in fibrotic processes: review of pharmacological agents and new approaches with amino acids and their analogues. J Invest Dermatol 1982;79:113s-120s. 2. Tan EML, Ryhlnen L, Uitto J. Proline analogues inhibit human skin fibroblast growth collagen production in culture. J Invest Dermatol 1983;80:261-7. 3. Riley DJ, Kerr JS, Berg RA, et al. Prevention of bleomycininduced pulmonary fibrosis in the hamster by cis-4-hydroxy-L-proline. Am Rev Respir Dis 1981;123:388-93. 4. Rojkind M. Inhibition of liver fibrosisby L-azetidine-2-carboxylic acid in rats treated with carbon tetrachloride.J Clin Invest 1973;52:2451-6. 5. Wiestner M, Krieg T, Dietrich H, Glanville RW, Fietzek P, Miiller PK. Inhibiting effect of procollagen peptides on collagen biosynthesis in fibroblast cultures. J Biol Chem 1979;254:7016-23. Wu CH, Donovan CB, Wu GY. Evidence for pretransla6. tional regulation of collagen synthesis by procollagen propeptides. J Biol Chem 1986;261:10482-4. 7. Wu CH, Walton CM, Wu GY. Propeptide-mediated regulation of procollagen synthesis in IMR-90 human lung fibroblast cell cultures. J Biol Chem 1991;266:2983-7. 8. Siegel RC. Collagen cross-linking:effect of D-penicillamine on cross-linking in vitro. J Biol Chem 1977;252:254-9. 9. Vater CA, Harris Jr ED, Siegel RC. Native cross-links in collagen fibrils induce resistance to human synovial collagenase. Biochem J 1979;181:639-45. 10. Asboe-Hansen G. Treatment of generalized scleroderma: updated results. Acta-Dermatovenereol1979;59:465 -467. 11. Moynahan EJ. Penicillamine in the treatment of morphoea and keloid in children. Postgrad Med J (Suppl); 1974;3940. 12. Berman B, Duncan MR. Pentoxifylline inhibits normal human dermal fibroblast in vitro proliferation, collagen, glycosaminoglycan, and fibronectin production, and increases collagenase activity. J Invest Dermatol 1989; 92~605-10. 13. Berman B, Duncan MR. Pentoxifylline inhibits the proliferation of human fibroblasts derived from keloid, scleroderma and morphoea skin and their production of collagen, glycosaminoglycans and fibronectin. Br J Dermatol 1990; 1231339-46. 14. Rubinstein M, Novick D, Fischer DG. The human interferon-y receptor system. Immunol Rev 1987;97:29-50. 15. Jimenez SA, Freundlich B, Rosenbloom J. Selective inhibition of human diploid fibroblast collagen synthesis by interferons. J Clin Invest 1984;74:1112-6. 16. Duncan MR,Berman B. y-Interferon is the lymphokine and /?-interferon the monokine responsible for inhibition of fibroblast collagen production and late but not early fibroblast proliferation. J Exp Med 1985;162:516-27.
17. Duncan MR, Berman B. Differential regulation of glycosaminoglycan, fibronectin, and collagenase production in cultured human dermal fibroblasts by interferon-alpha, -beta, and -gamma. Arch Dermatol Res 1989;281:11-8. 18. Duncan MR, Berman B. Persistence of a reduced-collagenproducing phenotype in cultured scleroderma fibroblasts after short-term exposure to interferons. J Clin Invest 1987;79:1318-24. 19. Granstein RD, Murphy GF, Margolis RJ, Byme MH, Amento EP. Gamma-interferon inhibits collagen synthesis in vivo in the mouse. J Clin Invest 1987;79:1254-8. 20. Kiihiri V-M, Heino J, Vuorio T, Vuorio E. Interferon-a and interferon-y reduce excessive collagen synthesis and procollagen mRNA levels of scleroderma fibroblasts in culture. Biochim Biophys Acta 1988;968:45-50. 21. Kahan A, Amor B, Menkes CJ, Strauch G. Recombinant interferon-y in the treatment of systemic sclerosis. Am J Med 1989;87273-7. 22. Granstein RD, Rook A, Flotte TJ, et al. Controlled trial of intralesional recombinant interferon-y in the treatment of keloidal scarring. Arch Dermatol 1990;126:1295-302. 23. Larrabee WF Jr, East CA, Jaffe HS, Stephenson C, Peterson KE. Intralesional interferon gamma treatment for keloids and hypertrophic scars. Arch Otolaryngol Head Neck Surg 1990;116:1159-62. 24. Berman 8, Duncan MR. Short-term keloid treatment in vivo with human interferon alfa-2b results in a selective and persistent normalization of keloidal fibroblast collagen, glycosaminoglycan, and collagenase production in vitro. J Am Acad Dermatol 1989;21:694-702. 25. Goslen JB. Wound healing after cosmetic surgery. In: Coleman 111 WP, Hanke CW, Alt TH, Asken S, eds. Cosmetic Surgery of the Skin Principles and Techniques. Philadelphia: BC Decker, 1991:47-63. 26. Pinnell SR. Regulation of collagen synthesis. J Invest Dermatol (suppl) 1982;79:73-6. 27. Murray JC, Pollack SV, Pinnell SR. Keloids: a review. J Am Acad Dermatol 1981;4:461-70. 28. Russo D, Fanin R, Zuffa E, et al. Treatment of Ph+ chronic myeloid leukemia by gamma interferon. Blut 1989;59:1520. 29. Maluish AE, Urba WJ, Longo DL, et al. The determination of an immunologically active dose of interferon-gamma in patients with melanoma. J Clin Oncol 1988;6:434-45. 30. Aulitzky WE,Aulitzky W, Gastl G, et al. Acute effects of single doses of recombinant interferon-y on blood cell counts and lymphocyte subsets in patients with advanced renal cell cancer. J Interferon Res 1989;9:425-33. 31. Roberts AB, Spom MB, Assoian RK, et al. Transforming growth factor type 8:rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci USA 1986;83:4167-71. 32. Ignotz RA,MassaguC J. Transforming growth factor-Bstimdates the expression of fibronectin and collagen and their
1Dermatol Surg Oncol 1992;18:981-986
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
incorporation into the extracellular matrix. J Biol Chem 1986;261:4337-45. Ignotz RA, Endo T, MassaguC J. Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor$. J Biol Chem 1987;262:6443-6. PeItonen J, Hsiao LL, Jaakkola S, et al. Activation of collagen gene expression in keloids co-localization of types I and VI collagen and transforminggrowth factor-@ mRNA. J Invest Dermatol 1991;97240-8. Gruschwitz M, Miiller PU, Sepp N, Hofer E, Fontana A, Wick G. Transcription and expression of transforming growth factor type beta in the skin of progmsive systemic sclerosis a mediator of fibrosis? J Invest Dermatol 1990; 94~197-203. Peltonen J, K5hiiri L, Jaakkola S, et al. Evaluation of transforming growth factor B and type I procollagen gene expression in fibrotic skin diseases by in situ hybridization. J Invest Dermatol 1990;94:365-71. Czaja MJ, Weiner FR, Flanders KC, et al. In vitro and in vivo association of transforming growth factor-fl with hepatic fibrosis. J Cell Biol 1989;108:2477-82. Border WA Okuda S, Languino LR, Spom MB,Ruoslahti E. Suppression of experiznental glomdonephritis by antiserum against transforminggrowth factor @. Nature 1990; 346371 -4. Assoian RK, Komoriya A, Meyers CA, Miller DM, Spom MB. Transforming growth factor-/? in human platelets: identification of a major storage site, purification and characterization.J Biol Chem 1983;258:7155-7160. Assoian RK, FleurdelysBE, StevensonHC, et al. Expression and secretion of type b transforming growth factor by activated human macrophages. Proc Natl Acad Sci USA 1987; 84:6020 -4. Kerhl JH,Wakefield LM, Roberts AB, et al. Production of transforminggrowth factors by human T lymphocytes and its potential role in the regulation of T cell growth. J Exp Med 1986;163:1037-50. Spom MB, Roberts AB. Transforming growth factor-fi multiple actions and potential clinical applications. JAMA 1989;262:938-41. Falanga V, Tiegs SL, Alstadt SP, Roberts AB, Spom MB. Transforming growth factor-beta: selective inaease in glycosaminoglycan synthesis by cultures of fibroblasts from patients with progressive systemic sclerosis. J Invest Dermatol 1987;89: 100-4. Spom MB, Roberts AB, Wakefield LM,de CrombruggheB. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol 1987; 105:1039 -45. Novick D, Engelmann H, Wallach D, Rubinstein M. Soluble cytokine receptorsare present in normal human urine. J Exp Med 1989;170:1409-14. H~~IIUXII CH, W ~ ~ CCJ, O XArend WP, et al. Interleukin-1 receptor antagonist activityof a human interleukin-1inhibitor. Nature 1990;343:336-40.
LOW AND MOY 985 COLLAGEN AND EXCESSIVE SCARRING
47. EisenbergSP, Evans RJ, Arend WP,et al. Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist. Nature 1990; 343~341-6. 48. Femandez-BotranR. Soluble cytokine receptors: their role in immunoregulation.FASEB 1991;5:2567-74. 49. Andres JL, Stanley K, Cheifetz S, MassaguC J. Membraneanchored and soluble forms of betaglycan, a polymorphic proteoglycan that binds transforming growth factor-j?. J Cell Biol1989;109:3 137- 45. 50. Rubin LA, Kurman CC, Fritz ME, et al. Solubleinterleukin 2 receptorsare released from activatedhuman lymphoid cells in vitro. J Immun01 1985;135:3172-7. 51. Treiger BF, Leonard WJ, Svetlik P, Rubin LA, Nelson DL, Greene WC. A secreted form of the human interleukin 2 receptor encoded by an “anchor minus” cDNA. J Immunol 1986;136:4099- 105. 52. Osawa H, Josimovic-Alasevic0, Diamantstein T. Interleukin 2 receptors are released by cells in vim and in vivo. I. Detection of soluble IL 2 receptors in cell culture supematants and in the serum of mice by an immunoradiometric m y . EW J Immunol 1986;16:467-9. 53. MosIey B, BeckmannMP,March CJ, et al. The murine interleukin-4 receptor: molecular cloning and characterization of secreted and membrane bound forms. Cell 1989; 59~335-48. 54. Peetre C, Thysell H, Grubb A, Olsson I. A tumor necrosis factor binding protein is present in human biological fluids. EUI J Haemat01 1988;41:414-9. 55. Engelmann H, Aderka D, Rubinstein M, Rotman D, Wallach D. A tumor necrosis factor-bindingprotein purified to homogeneity from human urine protects cells from tumor necrosis factor toxiaty. J Biol Chem 1989;264:11974-80. 56. Schall TJ, Lewis M, Koller KJ, et al. Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell 1990;61:361- 70. 57. Bendtzen K, Svenson M, JBnsson V, Hippe E. Autoantibodies to cytokines-friends or foes? Immunol Today 1990; 11:167-9. 58. Svenson M, Poulsen LK, Fomsgaard A, Bendtzen K. IgG autoantibodies against interleukin la in sera of normal ind i v i d d . Scand J Imm~11011989;29:489-92. 59. Bendtzen K, Svenson M, Fomsgaard A, Poulsen LK. Native inhibitors (autoantibodies) of IL-la and TNF. Immunol Today 1989;10:222. 60. Fomsgaard A, Svenson M, Bendtzen K. Auto-antibodies to lumour nec-rosis factor a in healthy humans and patients with inaammatory diseases and gram-negative bacterial infections. Scand J Immun011989;30:219-3. 61 Ross C, Hansen MB,Schyberg T, Berg K. Autoantibodiesto crude human leucocyte interferon (IFN), native human IFN, recombinant human IFN-alpha 2b and human IFNgamma in healthy blood donors. Clin Exp Immunol. 1990;82:57-62.
986 LOW AND MOY
62. O’Connor-McCourt MD, Wakefield LM. Latent transforming growth factor$ in serum: a specific complex with a,macroglobulin. J Biol Chem 1987;262: 14090- 9. 63. Huang SS, OGrady P, Huang JS. Human transforming growth factor /3 a,-macroglobulin complex is a latent form of transforming growth factor /3. J Biol Chem 1988;263:1535-41. 64. LaMarre J,Wollenberg GK, Gonias SL, Hayes MA. Cytokine binding and clearance properties of proteinase-activated a,-macroglobulins. Lab Invest 1991;65:3- 14. 65. Huang JS,Huang SS, Deuel TF. Specificcovalent binding of platelet-derived growth factor to human plasma az-macroglobulin. Proc Natl Acad Sci USA 1984;81:342-6. 66. LaMarre J,Wollenberg GK, Gonias SL, Hayes MA. Reaction of a,-macroglobulin with plasmin increases binding of transforming growth factors-fl and 82. Biochim Biophys Acta 1990;1091:197-204.
-
Dennatol Surg Oncol 1992;18:981-986 67. LaMarre J,Hayes MA, Wollenberg GK, Hussaini I, Hall SW, Gonias SL. An a,-macroglobulin receptor-dependent mechanism for the plasm clearance of transforming growth factor-/3l in mice. J Clin Invest 1991;8739-44. 68. Stimson WH.Quantitation of a new serum a-macroglobulin in malignant disease, steroid treatment, pregnancy and normal subjects. J Endocr 1974;61:30-31. 69. Stimson WH.Variations in the level of a pregnancy-assoaated a-macroglobulin in patients with cancer. J Clin Path 1975;28:868-71. 70. Yamaguchi Y, Mann DM, Ruoslahti E. Negative regulation of transforming growth factor-/3 by the proteoglycan decorin. Nature 1990;346:281-4. 71. MacKay K, Robbms AR, Bruce MD, Danielpour D. Identification of disulfide-linked transforming growth factor-/3l-speci6c binding proteins in rat glomeruli. J Biol Chem 1990;265:9351-6.
