Ultraviolet Irradiation Increases Matrix Metalloproteinase-8 Protein in Human Skin In Vivo
2001; Elsevier BV; Volume: 117; Issue: 2 Linguagem: Inglês
10.1046/j.0022-202x.2001.01432.x
ISSN1523-1747
AutoresGary J. Fisher, Hye-Jung Choi, Zsuzsanna Bata‐Csörgõ, Yuan Shao, Subhash C. Datta, Z.-Q. Wang, Sewon Kang, John J. Voorhees,
Tópico(s)Antioxidant Activity and Oxidative Stress
ResumoHumans express three distinct collagenases, MMP-1, MMP-8, and MMP-13, that initiate degradation of fibrillar type I collagen. We have previously reported that ultraviolet irradiation causes increased expression of MMP-1, but not MMP-13, in keratinocytes and fibroblasts in human skin in vivo. We report here that ultraviolet irradiation increases expression of MMP-8 in human skin in vivo. Western analysis revealed that levels of the full-length, 85 kDa proenzyme form of MMP-8 increased significantly within 8 h post ultraviolet irradiation (2 minimal erythema doses). Increased full-length MMP-8 protein was associated with infiltration into the skin of neutrophils, which are the major cell type that expresses MMP-8. Immunofluorescence revealed coexpression of MMP-8 and neutrophil elastase, a marker for neutrophils. Immunohistology demonstrated MMP-8 expression in neutrophils in the papillary dermis between 4 and 8 h post ultraviolet irradiation, and in the epidermis at 24 h post radiation. MMP-8 mRNA expression was not detected in nonirradiated or ultraviolet-irradiated human skin, indicating that increased MMP-8 following ultraviolet irradiation resulted from preexisting MMP-8 protein in infiltrating neutrophils. Pretreatment of skin with the glucocorticoid clobetasol, but not all-trans retinoic acid, significantly blocked ultraviolet-induced increases in MMP-8 protein levels, and neutrophil infiltration. In contrast, all-trans retinoic acid and clobetasol were equally effective in blocking ultraviolet induction of MMP-1 and degradation of collagen in human skin in vivo. Taken together, these data demonstrate that ultraviolet irradiation increases MMP-8 protein, which exists predominantly in a latent form within neutrophils, in human skin in vivo. Although ultraviolet irradiation induces both MMP-1 and MMP-8, ultraviolet-induced collagen degradation is initiated primarily by MMP-1, with little, if any, contribution by MMP-8. Humans express three distinct collagenases, MMP-1, MMP-8, and MMP-13, that initiate degradation of fibrillar type I collagen. We have previously reported that ultraviolet irradiation causes increased expression of MMP-1, but not MMP-13, in keratinocytes and fibroblasts in human skin in vivo. We report here that ultraviolet irradiation increases expression of MMP-8 in human skin in vivo. Western analysis revealed that levels of the full-length, 85 kDa proenzyme form of MMP-8 increased significantly within 8 h post ultraviolet irradiation (2 minimal erythema doses). Increased full-length MMP-8 protein was associated with infiltration into the skin of neutrophils, which are the major cell type that expresses MMP-8. Immunofluorescence revealed coexpression of MMP-8 and neutrophil elastase, a marker for neutrophils. Immunohistology demonstrated MMP-8 expression in neutrophils in the papillary dermis between 4 and 8 h post ultraviolet irradiation, and in the epidermis at 24 h post radiation. MMP-8 mRNA expression was not detected in nonirradiated or ultraviolet-irradiated human skin, indicating that increased MMP-8 following ultraviolet irradiation resulted from preexisting MMP-8 protein in infiltrating neutrophils. Pretreatment of skin with the glucocorticoid clobetasol, but not all-trans retinoic acid, significantly blocked ultraviolet-induced increases in MMP-8 protein levels, and neutrophil infiltration. In contrast, all-trans retinoic acid and clobetasol were equally effective in blocking ultraviolet induction of MMP-1 and degradation of collagen in human skin in vivo. Taken together, these data demonstrate that ultraviolet irradiation increases MMP-8 protein, which exists predominantly in a latent form within neutrophils, in human skin in vivo. Although ultraviolet irradiation induces both MMP-1 and MMP-8, ultraviolet-induced collagen degradation is initiated primarily by MMP-1, with little, if any, contribution by MMP-8. Photoaging, caused by repeated exposure to solar ultraviolet (UV) irradiation, results in both clinical and histologic changes in human skin (Havlik et al., 1999Havlik N.L. Fitzpatrick T.B. Kligman A.M. Kligman L.H. Geriatric dermatology.in: Freedberg I.M. Eisen A.Z. Wolff K. Austen K.F. Goldsmith L.A. Katz S.I. Fitzpatrick T.B. Fitzpatrick's Dermatology in General Medicine. 2. McGraw-Hill, New York1999: 1707-1723Google Scholar). Characteristic clinical features of photodamaged skin include both fine and coarse wrinkling, mottled pigmentation, dryness, and loss of skin tone (Gilchrest and Yaar, 1992Gilchrest B.A. Yaar M. Aging and photoaging of the skin: observations at the cellular and molecular level.Br J Dermatol. 1992; 127: 25-30Crossref PubMed Scopus (122) Google Scholar;Scharffetter-Kochanek, 1997Scharffetter-Kochanek K. Photoaging of the connective tissue of skin: its prevention and therapy.Adv Pharmacol. 1997; 38: 639-655Crossref PubMed Scopus (41) Google Scholar). Histologic and ultrastructural studies have revealed that the major alterations in photoaged skin are localized in the connective tissue (Scharffetter-Kochanek, 1997Scharffetter-Kochanek K. Photoaging of the connective tissue of skin: its prevention and therapy.Adv Pharmacol. 1997; 38: 639-655Crossref PubMed Scopus (41) Google Scholar), which is composed predominantly of collagen, elastin, proteoglycans, and fibronectin. As collagen fibrils and elastin are responsible for the strength and resilience of skin (Uitto, 1986Uitto J. Connective tissue biochemistry of the aging dermis: age-related alterations in collagen and elastin.Dermatol Clin. 1986; 4: 433-446PubMed Google Scholar), their degeneration with photoaging causes skin to become less youthful in appearance. Biochemical, quantitative, and qualitative changes have been reported in the dermal extracellular proteins elastin (Braverman and Fonferko, 1982Braverman I.M. Fonferko E. Studies in cutaneous aging: I. The elastic fiber network.J Invest Dermatol. 1982; 78: 434-443Crossref PubMed Scopus (375) Google Scholar;Uitto, 1986Uitto J. Connective tissue biochemistry of the aging dermis: age-related alterations in collagen and elastin.Dermatol Clin. 1986; 4: 433-446PubMed Google Scholar), interstitial collagen (Trautinger et al., 1989Trautinger F. Trenz A. Raff M. Kokoschka E. Influence of UV radiation on dermal collagen content in hairless mice.Arch Dermatol Res. 1989; 281: 144Google Scholar;Schwartz et al., 1991Schwartz E. Cruickshank F.A. Mezick J.A. Kligman L.H. Topical all-trans retinoic acid stimulates collagen synthesis in vivo.J Invest Dermatol. 1991; 96: 975-978Abstract Full Text PDF PubMed Google Scholar), and glycosaminoglycans (Sams and Smith, 1961Sams W.M. Smith J.G. The histochemistry of chronically sun-damaged skin.J Invest Dermatol. 1961; 37: 447-452Crossref PubMed Scopus (77) Google Scholar;Smith et al., 1962Smith J.G. Davidson E.A. Sams W.M. Clark R.D. Alterations in human dermal connective tissue with age and chronic sun damage.J Invest Dermatol. 1962; 39: 347-350Crossref PubMed Scopus (233) Google Scholar) in photoaged skin. Histologically, connective tissue damage induced by UV irradiation is primarily manifested as the disorganization of collagen fibrils (Bernstein et al., 1996Bernstein E.F. Chen Y.Q. Kopp J.B. Long-term sun exposure alters the collagen of the papillary dermis: comparison of sun-protected and photoaged skin by Northern analysis, immunohistochemical staining, and confocal laser scanning microscopy.J Am Acad Dermatol. 1996; 34: 209-218Abstract Full Text PDF PubMed Scopus (168) Google Scholar) that constitute the bulk (90% dry weight) of skin connective tissue, and accumulation of abnormal, amorphous, elastin-containing material (Lavker, 1995Lavker R. Cutaneous aging: chronologic versus photoaging.in: Gilchrest B.A. Photoaging. Blackwell Science, Cambridge. MA1995: 123-135Google Scholar). The matrix metalloproteinases (MMPs) are a family of enzymes responsible for degrading connective tissue (Murphy et al., 1990Murphy G. Hembry R.M. Hughes C.E. Fosang A.J. Hardingham T.E. Role and regulation of metalloproteinases in connective tissue turnover.Biochem Soc Trans. 1990; 18: 812-815Crossref PubMed Scopus (78) Google Scholar;Woessner, 1994Woessner J.F. The family of matrix metalloproteinases.Ann N Y Acad Sci. 1994; 732: 11-21Crossref PubMed Scopus (426) Google Scholar). They are structurally related endopeptidases that mediate degradation of different macromolecular components of the extracelluar matrix and the basement membrane (Matrisian, 1990Matrisian L.M. Metalloproteinases and their inhibitors in matrix remodeling.Trends Genet. 1990; 6: 121-125Abstract Full Text PDF PubMed Scopus (1505) Google Scholar;Woessner, 1991Woessner J.F. Matrix metalloproteinases and their inhibitors in connective tissue remodeling.FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3007) Google Scholar). The human family of MMPs is composed of at least 16 members, which can be classified into four different subfamilies; the collagenases, the gelatinases, the stromelysins, and the membrane MMPs (Shingleton et al., 1996Shingleton W.D. Hodges D.J. Brick P. Cawston T.E. Collagenase a key enzyme in collagen turnover.Biochem Cell Biol. 1996; 74: 759-775Crossref PubMed Scopus (124) Google Scholar;Kahari and Saarialho-Kere, 1997Kahari V.-M. Saarialho-Kere U. Matrix metalloproteinase in skin [review].Exp Dermatol. 1997; 6: 199-213Crossref PubMed Scopus (488) Google Scholar). In humans, there are three distinct collagenases: MMP-1 (collagenase 1 or interstitial collagenase), MMP-8 (neutrophil collagenase or collagenase 2), and MMP-13 (collagenase 3) (Shingleton et al., 1996Shingleton W.D. Hodges D.J. Brick P. Cawston T.E. Collagenase a key enzyme in collagen turnover.Biochem Cell Biol. 1996; 74: 759-775Crossref PubMed Scopus (124) Google Scholar;Vincenti et al., 1996Vincenti M.P. White L.A. Schroen D.J. Benbow U. Brinckerhoff C.E. Regulating expression of the gene for matrix metalloproteinase-1 (collagenase): mechanisms that control enzyme activity, transcription, and mRNA stability.Crit Rev Eukaryotic Gene Expression. 1996; 6: 391-411Crossref PubMed Scopus (236) Google Scholar). MMP-1 is expressed by keratinocytes and fibroblasts and functions in normal collagen turnover and matrix remodeling during wound healing. MMP-8 is expressed predominantly by neutrophils and released during inflammatory processes (Hasty et al., 1990Hasty K.A. Pourmotabbed T.F. Goldberg G.I. Thompson J.P. Spinella D.G. Stevens R.M. Mainardi C.L. Human neutrophil collagenase: a distinct gene product with homology to other matrix metalloproteinases.J Biol Chem. 1990; 265: 11421-11424Abstract Full Text PDF PubMed Google Scholar;Devarajan et al., 1991Devarajan P. Mookhtiar K. Van Wart H. Berliner N. Structure and expression of the cDNA encoding human neutrophil collagenase.Blood. 1991; 77: 2731-2738Crossref PubMed Google Scholar). MMP-13 is expressed in various epithelial cancers but, unlike MMP-1, it not expressed by either keratinocytes or fibroblasts during wound repair. MMP-13, however, is expressed in fibroblasts in chronic cutaneous ulcers (Ravanti et al., 1999Ravanti L. Heino J. Lopez-Otin C. Kahari V.M. Induction of collagenase-3 (MMP-13) expression in human skin fibroblasts by three-dimensional collagen is mediated by p38 mitogen-activated protein kinase.J Biol Chem. 1999; 274: 2446-2455Crossref PubMed Scopus (250) Google Scholar). There is significant homology between MMP-8 and MMP-1: MMP-8 exhibits 57% identity to the protein sequence for MMP-1 (Hasty et al., 1990Hasty K.A. Pourmotabbed T.F. Goldberg G.I. Thompson J.P. Spinella D.G. Stevens R.M. Mainardi C.L. Human neutrophil collagenase: a distinct gene product with homology to other matrix metalloproteinases.J Biol Chem. 1990; 265: 11421-11424Abstract Full Text PDF PubMed Google Scholar). MMP-8 cleaves type I collagen faster than type III collagen, whereas MMP-1 shows greater selectivity for type III collagen relative to type I collagen (Hasty et al., 1987Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. The collagen substrate specificity of human neutrophil collagenase.J Biol Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar;Hirose et al., 1993Hirose T. Patterson C. Pourmotabbed T. Mainardi C.L. Hasty K.A. Structure-function relationship of human neutrophil collagenase: identification of regions responsible for substrate specificity and general proteinase activity.Proc Natl Acad Sci. 1993; 90: 2569-2573Crossref PubMed Scopus (122) Google Scholar;Knauper et al., 1997Knauper V. Docherty A.J.P. Smith B. Tschesche H. Murphy G. Analysis of the contribution of the hinge region of human neutrophil collagenase (HNC, MMP-8) to stability and collagenolytic activity by alanine scanning mutagensis.FEBS Lett. 1997; 405: 60-64Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Both MMP-1 and MMP-8 are synthesized as latent proenzymes that require proteolytic processing to become catalytically active. Whereas MMP-1 is synthesized and released from cells into the extracelluar matrix, however, MMP-8 is synthesized and stored in specific granules in neutrophil leukocytes (Hasty et al., 1986Hasty K.A. Hibbs M.S. Kang A.H. Mainardi C.L. Secreted forms of human neutrophil collagenase.J Biol Chem. 1986; 261: 5645-5650Abstract Full Text PDF PubMed Google Scholar;Knauper et al., 1990Knauper V. Kramer S. Reinke H. Tschesche H. Characterization and activation of procollagenase from human polymorphonuclear leucocytes.Eur J Biochem. 1990; 189: 295-300Crossref PubMed Scopus (77) Google Scholar;Mallya et al., 1990Mallya S.K. Mookhtiar K.A. Gao Y. Brew K. Dioszegi M. Birkedal-Hansen H. van Wart H.E. Characterization of 58-kilodalton human neutrophil collagenase: comparison with human fibroblast collagenase.Biochemistry. 1990; 29: 10628-10634Crossref PubMed Scopus (70) Google Scholar). MMP-8 activity is therefore regulated by factors such as surface-bound ligands (IgG or complement components) that release it through degranulation (Chatham et al., 1990Chatham W. Heck L. Blackburn W.J. Ligand-dependent release of active neutrophil collagenase.Arthritis Rheum. 1990; 33: 228-234Crossref PubMed Scopus (16) Google Scholar). Once released and activated through proteolytic or oxidative mechanisms (Weiss, 1989Weiss S.J. Tissue destruction by neutrophils.New Engl J Med. 1989; 320: 365-375Crossref PubMed Scopus (3745) Google Scholar), MMP-8 plays a major role in the connective tissue turnover that accompanies inflammatory processes (Weiss et al., 1985Weiss S.J. Peppin G. Ortiz X. Ragsdale C. Test S.T. Oxidative autoactivation of latent collagenase by human neutrophils.Science. 1985; 227: 747-749Crossref PubMed Scopus (336) Google Scholar;Knauper et al., 1990Knauper V. Kramer S. Reinke H. Tschesche H. Characterization and activation of procollagenase from human polymorphonuclear leucocytes.Eur J Biochem. 1990; 189: 295-300Crossref PubMed Scopus (77) Google Scholar;Claesson et al., 1996Claesson R. Karlsson M. Zhang Y.-Y. Carlsson J. Relative role of chloramines, hypochlorous acid, and protease in the activation of human polymorphonuclear leukocyte collagenase.J Leukoc Biol. 1996; 60: 598-602PubMed Google Scholar;Okamoto et al., 1997Okamoto T. Akaike T. Nagano T. et al.Activation of human neutrophil procollagense by nitrogen dioxide and peroxynitrite: a novel mechanism for procollagenase activation involving nitric oxide.Arch Biochem Biophys. 1997; 342: 261-274https://doi.org/10.1006/abbi.1997.0127Crossref PubMed Scopus (193) Google Scholar). UV irradiation induces MMP-1, but not MMP-13, in human skin in vivo. In addition, UV irradiation induces 92 kDa gelatinase (MMP-9) and stromelysin-1 (MMP-3) in human skin in vivo (Fisher et al., 1996Fisher G.J. Datta S.C. Talwar H.S. Wang Z.Q. Varani J. Kang S. Voorhees J.J. Molecular basis of sun-induced premature skin ageing and retinoid antagonism.Nature. 1996; 379: 335-339Crossref PubMed Scopus (1119) Google Scholar). Together, these three MMPs can fully degrade skin collagen (Matrisian and Hogan, 1990Matrisian L.M. Hogan B.L. Growth factor-regulated proteases and extracellular matrix remodeling during mammalian development.Curr Top Dev Biol. 1990; 24: 219-259Crossref PubMed Scopus (195) Google Scholar;Birkedal-Hansen et al., 1993Birkedal-Hansen H. Moore W.G. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Matrix metalloproteinases: a review.Crit Rev Oral Biol Med. 1993; 329: 530-535Google Scholar). Initially, MMP-1 cleaves the triple-helical collagen molecule into three-quarter and one-quarter length fragments. Collagen is then further degraded into smaller fragments by MMP-3 and MMP-9 (Hasty et al., 1990Hasty K.A. Pourmotabbed T.F. Goldberg G.I. Thompson J.P. Spinella D.G. Stevens R.M. Mainardi C.L. Human neutrophil collagenase: a distinct gene product with homology to other matrix metalloproteinases.J Biol Chem. 1990; 265: 11421-11424Abstract Full Text PDF PubMed Google Scholar;Shingleton et al., 1996Shingleton W.D. Hodges D.J. Brick P. Cawston T.E. Collagenase a key enzyme in collagen turnover.Biochem Cell Biol. 1996; 74: 759-775Crossref PubMed Scopus (124) Google Scholar;Kahari and Saarialho-Kere, 1997Kahari V.-M. Saarialho-Kere U. Matrix metalloproteinase in skin [review].Exp Dermatol. 1997; 6: 199-213Crossref PubMed Scopus (488) Google Scholar). MMPs are likely to be the primary mediators of connective tissue damage in skin exposed to UV irradiation (Fisher et al., 1997Fisher G.J. Wang Z.Q. Datta S.C. Varani J. Kang S. Voorhees J.J. Pathophysiology of premature skin aging induced by ultraviolet light.N Engl J Med. 1997; 337: 1419-1428Crossref PubMed Scopus (1080) Google Scholar). To better understand the mechanism of skin collagen breakdown associated with the photoaging process, we have investigated whether UV irradiation alters MMP-8 expression in human skin in vivo. We have also examined the effects of two different classes of compounds, all-trans retinoic acid and the glucocorticoid clobetasol, which inhibit UV induction of MMP-1, on expression of MMP-8 in human skin in vivo. Sun-protected buttock skin of adult Caucasian subjects, who were without current or prior skin disease, was pretreated with 0.1% all-trans retinoic acid, 0.05% clobetasol propionate, or its vehicle (70% ethanol, 30% propylene glycol, and 0.05% butylated hydroxytoluene) for 24 h under occlusion prior to UV exposure. Sun-protected buttock skin of subjects was exposed to twice the minimal erythema dose (2MED) of UV. Two UV sources were utilized: (i) four F36T12 ERE-VHO UVB tubes filtered with Kodacel to remove wavelengths below 290 nm (Fisher et al., 1997Fisher G.J. Wang Z.Q. Datta S.C. Varani J. Kang S. Voorhees J.J. Pathophysiology of premature skin aging induced by ultraviolet light.N Engl J Med. 1997; 337: 1419-1428Crossref PubMed Scopus (1080) Google Scholar); and (ii) a 450 W xenon arc solar simulator filtered with a Schott WG320 filter to remove wavelengths below 290 nm, coupled to a liquid light guide. The spectral output of these two sources was determined with an OL754 spectroradiometer (Optronic Laboratories, Orlando, FL). The UV spectrum (290–400 nm) for UVB was composed of 0.3% UVC, 65.1% UVB, 24.4% UVA2, and 10.2% UVA1. The UV spectrum for the xenon arc lamp was 0.00006% UVC, 6.6% UVB, 16.5% UVA2, and 76.8% UVA1. Light output was monitored with an IL 4443 phototherapy radiometer and a SED240/UVB/UV photodetector (International Light, Newbury, MA). For studies with multiple time points, tissue was obtained from each subject at each time point. Replicate 4 mm or 6 mm punch biopsies of skin were obtained from irradiated and adjacent nonirradiated sites. Skin samples for enzyme-linked immunosorbent assay (ELISA), Western analysis, and reverse transcriptase polymerase chain reaction (RT-PCR) were snap-frozen in liquid nitrogen and stored at -70°C. Skin samples for immunohistology were oriented in OCT embedding compound (Miles Laboratories, Elkhart, IN) prior to snap freezing. The University of Michigan Institutional Review Board approved all procedures involving human subjects, and all subjects provided written informed consent. Seven millimeter frozen sections were fixed in 2% paraformaldehyde and endogenous peroxidase activity was quenched with 0.3% H2O2. Sections were stained with a rabbit polyclonal antibody against MMP-8 (Chemicon, Temecula, CA) and a mouse monoclonal IgG1 antibody against neutrophil elastase (Dako, Glostrup, Denmark). Binding of the anti-MMP-8 antibody was visualized by biotinylated goat antirabbit (Vector, Burlingame, CA) in combination with the TSA Fluorescence System (Perkin Elmer-NEN Life Technologies, Boston, MA). The neutrophil elastase antibody was detected with goat antimouse IgG Texas Red Conjugate (CalBiochem, San Diego, CA). Stained sections were photographed with a Spot 2 Cooled Color Digital Camera (Diagnostic Instruments, Sterling Heights, MI) using a Zeiss Axioskop 2 microscope (Zeiss, Thornwood, NY). Immunohistologic analysis of MMP-8 was performed as described previously (Griffiths et al., 1993Griffiths C.E.M. Russman A.N. Majmudar G. Singer R.S. Hamilton T.A. Voorhees J.J. Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid).N Engl J Med. 1993; 329: 530-535Crossref PubMed Scopus (377) Google Scholar), with the follow ing modifications. Frozen sections were fixed in 2% paraformaldehyde and permeabilized with 0.1% Triton X-100. MMP-8 protein was detected with mouse monoclonal IgG1 antibody (Oncogene Research Products, Cambridge, MA). Neutrophil elastase was detected with a mouse monoclonal IgG1 antibody (Dako). Appropriately diluted mouse IgG1 was used as control. MMP-8 and MMP-1 proteins were assayed with the Biotrak capture ELISA (Amersham Pharmacia, Arlington Heights, IL). For determining MMP-8 protein levels, skin samples were homogenized by vigorous vortexing with 2.5 mm glass beads (Biospec, Bartlesville, OK) in 20 mM Tris (pH 7.6), 5 mM CaCl2, containing protease inhibitor mixture. The homogenate was centrifuged at 10,000 × g for 10 min and the supernatant was collected for analyses. MMP-1 protein was assayed from supernatant obtained from skin biopsies that had been incubated in Dulbecco's modified Eagle's medium (Life Technologies, Rockville, MD) for 8 h at 37°C. Protein content was determined by Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA), using bovine serum albumin as standard. MMP-8 protein levels in supernatants from skin homogenates, prepared as described above for ELISA analysis, were determined by Western blot analysis, as described previously (Fisher et al., 1996Fisher G.J. Datta S.C. Talwar H.S. Wang Z.Q. Varani J. Kang S. Voorhees J.J. Molecular basis of sun-induced premature skin ageing and retinoid antagonism.Nature. 1996; 379: 335-339Crossref PubMed Scopus (1119) Google Scholar). Equal amounts of protein (100 µg per lane) were analyzed for each treatment group. Immunoreactive MMP-8 protein was detected by enhanced chemifluorescence (Amersham Pharmacia) using a monoclonal antibody against human MMP-8 that detects both the full-length latent (85 kDa) and active (64 kDa) forms (Matsuki et al., 1996Matsuki H. Fujimoto N. Iwata K. Knauper V. Okada Y. Hayakawa T. A one-step sandwich enzyme immunoassay for human matrix metalloproteinase 8 (neutrophil collagnase) using monoclonal antibodies.Clin Chim Acta. 1996; 244: 129-143Crossref PubMed Scopus (40) Google Scholar). Immunoblots were visualized and quantified by STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Total RNA from human skin, primary cultured human keratinocytes, and fibroblasts was purified by guanidine-thiocyanate extraction (Stratagene, La Jolla, CA). MMP-8 mRNA expression was assessed by RT-PCR analysis. Briefly, cDNA was prepared using a constant amount of total cellular RNA (1 µg), reverse transcriptase, and random oligo primers. The cDNA was amplified in the presence of specific primers for human MMP-8 (5′-AGC TGT CAG AGG CTG AGG TAG AAA G and 5′-CCT GAA AGC ATA GTT GGG ATA CAT). Amplification conditions were denaturation for 40 s at 94°C, annealing for 60 s at 55°C, and extension for 90 s at 72°C for 33 cycles. RT-PCR amplification of the mRNA for the ribosomal protein 36B4 served as internal control. Reaction products were subjected to electrophoresis on 1.5% agarose gel, visualized with Vistra Green (Molecular Probes, Eugene, OR), and quantified by STORM PhosphorImager (Molecular Dynamics). Partially degraded collagen in the dermis of nonirradiated and UV-irradiated human skin was quantified by modifications of the procedure described byBank et al., 1997Bank R. Krikken M. Beckman B. Stoop R. Maroudas A. Lafeber F. Koppele J. A simplified measurement of degraded collagen in tissue: applications in healthy, fibrotic and osteoarthritic collagen.Matrix Biol. 1997; 16: 233-243Crossref PubMed Scopus (161) Google Scholar. This assay is based on the ability of α-chymotrypsin to extensively hydrolyze partially degraded, but not intact, native insoluble fibrillar collagen. The soluble proteolytic fragments derived from collagen are quantified by hydroxyproline content. Full-thickness skin samples were homogenized in 20 mM Tris HCl (pH 7.3) containing 1 mM iodoacetamide, 1 mM ethylenediamine tetraacetic acid, and pepstatin A (10 µg per ml) and centrifuged at 50 000 rpm for 40 min. Pellets were washed three times with 5 mM Tris (pH 7.3) and centrifuged at 10,000 × g for 5 min. α-Chymotrypsin was added to the pellets (75 µg in 200 µl in 5 mM Tris) and incubated at 37°C for 8 h. Reaction mixtures were centrifuged at 10,000 × g for 10 min, and proteolytic fragments released into supernatants were hydrolyzed with 6 N HCl at 110°C for 24 h. Hydrolysates were dried under vacuum, dissolved in distilled water, and redried under vacuum to remove traces of HCl. Finally, samples were dissolved in 0.1 M borate buffer (pH 9.5) and derivatized with o-phthaldialdehyde and 9-fluorenylmethyl chloroformate according to the procedure ofBank et al., 1997Bank R. Krikken M. Beckman B. Stoop R. Maroudas A. Lafeber F. Koppele J. A simplified measurement of degraded collagen in tissue: applications in healthy, fibrotic and osteoarthritic collagen.Matrix Biol. 1997; 16: 233-243Crossref PubMed Scopus (161) Google Scholar. Derivatized hydroxyproline, derived from collagen, was quantified by high performance liquid chromatography using an analytical C-18 Econosphere column. Isocratic elution of hydroxyproline was performed with 20 mM citric acid containing 5 mM tetramethylammonium chloride (pH 2.85) and 38% acetonitrile, at a flow rate of 1 ml per min. Derivatized hydroxyproline was measured by fluorescence at 330 nm (excitation wavelength 254 nm), using a Hitachi (Model 1080) fluorescence detector. Time course data, and data from comparisons of vehicle-treated skin to skin pretreated with all-trans retinoic acid or clobetasol, were analyzed with paired t tests. All p-values are two-tailed, and differences were considered significant for p < 0.05. MMP-8 protein was minimally detectable by Western blot analysis in extracts from nonirradiated human skin Figure 1a. MMP-8 protein levels were increased approximately 4-fold within 8 h and remained elevated for 24 h after UV irradiation Figure 1a. By 48 h post-UV, MMP-8 protein levels returned to near-baseline levels. Western analyses detected the full-length 85 kDa latent form of MMP-8. The processed 64 kDa active form of MMP-8 was not detectable. MMP-8 protein levels in human skin extracts were quantified by ELISA. In nonirradiated human skin, MMP-8 protein levels were less than 0.5 ng per mg tissue homogenate protein. MMP-8 protein levels were increased to 5.4 ng per mg protein within 8 h following UV irradiation Figure 1b. MMP-8 protein levels remained elevated for 24 h, consistent with the western analysis described above Figure 1a. We next performed RT-PCR to determine whether UV increased MMP-8 mRNA. MMP-8 mRNA was not detected in either nonirradiated or UV-irradiated human skin in vivo. Additionally, no MMP-8 mRNA was detect by RT-PCR in nonirradiated or UV-irradiated cultured human keratinocytes or dermal fibroblasts. Control MMP-8 cDNA was reproducibly amplified, indicating that lack of detection of MMP-8 mRNA in skin was not due to failure of the PCR amplification. In addition, 36B4 mRNA was amplified in all skin samples examined (N = 10, data not shown), indicating that RNA preparations were suitable for the RT-PCR analysis. The above data suggest that preformed MMP-8 protein is brought into the skin following UV irradiation. To examine this issue, we utilized immunohistology to localize MMP-8 protein in human skin. Immunohistology revealed increased expression of MMP-8 protein in cells in the dermis within 8 h post-UV (Figure 2a, b). Cells expressing MMP-8 protein were prominently localized near blood vessels in the dermis. MMP-8-positive cells were detected in both papillary dermis and epidermis by 24 h post-UV Figure 2c. At 48–72 h post-UV, MMP-8-positive cells were minimally detectable (data not shown), as was observed in non-UV-irradiated skin. The above data demonstrate that UV irradiation induces influx into skin of cells expressing MMP-8 protein. Neutrophils express MMP-8 and are known to infiltrate skin following UV irradiation (Hawk et al., 1988Hawk J.L.M. Murphy G.M. Holden C.A. The presence of neutrophils in human cutaneous ultraviolet-B inflammation.Br J Dermatol. 1988; 118: 27-30Crossref PubMed Scopus (89) Google Scholar;Strickland et al., 1997Strickland I. Rhodes L.E. Flanagan B.F. Friedman P.S. TNF-α and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression.J Invest Dermatol. 1997; 108: 763-768Crossref PubMed Scopus (158) Google Scholar). To determine whether these MMP-8-expressing cells were neutrophils, we performed immunohistology for neutrophil elastase, a marker
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