Matrix Metalloproteinase-8 Is Expressed in Rheumatoid Synovial Fibroblasts and Endothelial Cells

Abstract

Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane. In addition, we detected MMP-8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-α (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nm). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 μm doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis. Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane. In addition, we detected MMP-8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-α (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nm). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 μm doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis. Extracellular matrix degradation is fundamental to connective tissue remodeling during physiological processes as well as during the progress of several pathological phenomena. Matrix turnover is regulated by a delicate balance among the production, activation, and inhibition of proteolytic enzymes. The matrix metalloproteinases (MMPs) 1The abbreviations used are: MMP(s), matrix metalloproteinase(s); PMN, polymorphonuclear neutrophil(s) RSF, rheumatoid synovial fibroblasts; TNF-α, tumor necrosis factor-α; APMA, 4-aminophenylmercuric acetate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); DTPA, diethylenetriaminepentaacetic acid; kb, kilobase(s).1The abbreviations used are: MMP(s), matrix metalloproteinase(s); PMN, polymorphonuclear neutrophil(s) RSF, rheumatoid synovial fibroblasts; TNF-α, tumor necrosis factor-α; APMA, 4-aminophenylmercuric acetate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HUVEC, human umbilical vein endothelial cells; PBS, phosphate-buffered saline; RT, reverse transcription; PCR, polymerase chain reaction; bp, base pair(s); DTPA, diethylenetriaminepentaacetic acid; kb, kilobase(s). form a gene family of at least 14 enzymes participating in extracellular matrix remodeling. MMPs, together with the factors associated with their regulation, are reported to be highly implicated in various diseases such as rheumatoid arthritis, osteoarthritis, corneal ulceration, atherosclerosis, and tumor invasion and metastasis (for reviews, see Refs. 1Woessner J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3087) Google Scholar, 2Matrisian L.M. BioEssays. 1992; 14: 455-463Crossref PubMed Scopus (1329) Google Scholar, 3Murphy G. Docherty A.J.P. Am. J. Respir. Cell Mol. Biol. 1992; 7: 120-125Crossref PubMed Scopus (543) Google Scholar). Previous studies have demonstrated that neutrophil-derived MMPs such as collagenase (MMP-8) and gelatinase B (MMP-9, 92-kDa type IV collagenase), play a key role in the degradation of extracellular matrix constituents i.e. during the course of inflammatory diseases (4Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3847) Google Scholar, 5Konttinen Y.T. Lindy O. Suomalainen K. Ritchlin C. Saari H. Vauhkonen M. Lauhio A. Santavirta S. Sorsa T. Matrix. 1991; 11: 395-403Crossref PubMed Scopus (30) Google Scholar, 6Tschesche H. Bakowski B. Shettler A. Knäuper V. Reinke H. Biomed. Biochem. Acta. 1991; 50: 755-761PubMed Google Scholar, 7Sorsa T. Ding Y. Salo T. Lauhio A. Teronen O. Ingman T. Ohtani H. Andoh N. Takeha S. Konttinen Y.T. Ann. N. Y. Acad. Sci. 1994; 732: 112-131Crossref PubMed Scopus (143) Google Scholar). Collagenases exist as three distinct molecules, namely the fibroblast type (MMP-1, collagenase-1) (8Goldberg G.I. Wilhelm S.M. Kronberger A. Bauer E.A. Grant G.A. Eisen A.Z. J. Biol. Chem. 1986; 261: 6600-6605Abstract Full Text PDF PubMed Google Scholar), the neutrophil type (MMP-8) (9Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar), and collagenase-3 (MMP-13) (10Freije J.M. Diez-Itza I. Balbin M. Sanchez L.M. Blasco R. Tolivia J. Lopez-Otin C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar). They all are able to degrade specifically the fibrillar collagen types I, II, and III as well as type VII and X collagens (11Schmid T.M. Mayne R. Jeffrey J.J. Linsenmayer T.F. J. Biol. Chem. 1986; 261: 4184-4189Abstract Full Text PDF PubMed Google Scholar, 12Seltzer J.L. Adams S.A. Grant G.A. Eisen A.Z. J. Biol. Chem. 1981; 256: 4662-4668Abstract Full Text PDF PubMed Google Scholar), serpins (4Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3847) Google Scholar, 13Michaelis J. Vissers M.C.M. Winternbourn C.C. Biochem. J. 1990; 270: 809-814Crossref PubMed Scopus (71) Google Scholar), β-casein, and human α2-macroglobulin (14Sottrup-Jensen L. Birkedal-Hansen H. J. Biol. Chem. 1989; 264: 393-401Abstract Full Text PDF PubMed Google Scholar). Among collagenases, MMP-8 most effectively hydrolyzes the native type I and II collagens, whereas MMP-1 prefers type III collagen. MMP-8 is a considerably more efficient enzyme than MMP-1 with respect to almost all substrates except for type III collagen (9Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar). MMP-1 is transcribed and expressed by human fibroblasts, keratinocytes, endothelial cells, monocytes, and macrophages, and collagenase-3 by some human malignant breast tumors (10Freije J.M. Diez-Itza I. Balbin M. Sanchez L.M. Blasco R. Tolivia J. Lopez-Otin C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar), whereas MMP-8 has been shown to be stored in subcellular specific granules of mature human peripheral blood PMN after synthesis during PMN maturation in bone marrow (15Mainardi C.L. Pourmotabbed T.F. Hasty K.A. Am. J. Med. Sci. 1991; 302: 171-175Crossref PubMed Scopus (30) Google Scholar). Previous studies indicate that PMN MMP-8 transcription is completed before PMN emigrate from bone marrow (15Mainardi C.L. Pourmotabbed T.F. Hasty K.A. Am. J. Med. Sci. 1991; 302: 171-175Crossref PubMed Scopus (30) Google Scholar) and that MMP-8 activity is regulated by factors that affect the release of MMP-8 by degranulation and not its biosynthesis (4Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3847) Google Scholar). However, by using in situ hybridization, Cole and Kuettner (16Cole A.A. Kuettner K.E. Acta Orthop. Scand. 1995; 26: 98-102Crossref Scopus (39) Google Scholar) have shown that MMP-8 mRNA is also found in peripheral PMN. More recent observations (17Chubinskaya S. Huch K. Mikecz K. Cs-Szabo G. Hasty K.A. Kuettner K.E. Cole A.A. Lab. Invest. 1996; 74: 232-240PubMed Google Scholar, 18Cole A.A. Chubinskaya S. Schumacher B. Huch K. Cs-Szabo G. Yao J. Mikecz K. Hasty K.A. Kuettner K.E. J. Biol. Chem. 1996; 271: 11023-11026Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) showed MMP-8 mRNA expression in human cartilage, primarily in chondrocytes. In this study we investigated the expression and regulation of MMP-8, particularly in human rheumatoid synovial fibroblasts (RSF) and endothelial cells. It was shown that MMP-8 is also expressed in cell types other than those belonging to the PMN lineage. In these cells MMP-8 is secreted as a nonglycosylated protein. The expression of MMP-8 in RSF and endothelial cells was up-regulated by tumor necrosis factor-α (TNF-α) and phorbol 12-myristate 13-acetate (PMA). Doxycycline inhibited not only the catalytic activity of MMP-8, but also the expression of MMP-8 mRNA and protein. Medium 199 supplemented with 20 mmHepes was purchased from Flow Laboratories (Irvine, Scotland, U. K.). Ham's nutrient mixture F-12 was from Northumbria Biologicals Ltd. (Cramlington, Northumberland, U. K.). Dulbecco's modified Eagle's medium, keratinocyte-SFM, as well as other cell culture reagents were obtained from Life Technologies, Inc. 4-Aminophenylmercuric acetate (APMA), CHAPS, PMA, endoglycosidase F, N-glycosidase F, polyoxyethylene 23 lauryl ether (Brij-35), and doxycycline were purchased from Sigma. Native type I and II collagen were isolated from bovine skin and articular cartilage and analyzed for purity by cyanogen bromide cleavage peptide analysis (5Konttinen Y.T. Lindy O. Suomalainen K. Ritchlin C. Saari H. Vauhkonen M. Lauhio A. Santavirta S. Sorsa T. Matrix. 1991; 11: 395-403Crossref PubMed Scopus (30) Google Scholar). Human recombinant TNF-α (specific activity 2.45 × 107 units/mg) was a gift from J. Tavenier (Biogent, Gent, Belgium). Hydrocortisone was from Diosynth, (Oss, The Netherlands). The avidin-biotin-horseradish peroxidase kit and peroxidase-anti-peroxidase (rabbit) were from Dako A/S (DK-2600 Glostrup, Denmark). RSF were isolated from freshly dispersed tissue of patients with rheumatoid arthritis as described by Unemori et al. (19Unemori E.N. Hibbs M.S. J. Invest. 1991; PubMed Scopus Google Scholar). was were obtained by treatment at a of The cells were used after or Conditioned media were obtained by the cells in for with of 199 supplemented with human 50 and 50 to which the of was (10 TNF-α or The media were for at in a to cells and and were at Human umbilical vein endothelial cells were isolated and cultured as described A.A. J. Invest. PubMed Scopus Google Scholar, R. L. Biochem. J. PubMed Scopus Google Scholar). fibroblasts were from from the of during on and cultured as described T. Birkedal-Hansen H. H. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). Human fibroblasts were obtained and cultured by a described by and 1987; PubMed Scopus Google Scholar). Human were cultured as described in by al. T. Birkedal-Hansen H. H. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). Human cell cells were cultured as described M. A. K. Salo T. J. 1996; PubMed Scopus Google Scholar). marrow were obtained from patients being on for PMN were isolated and from blood of A. Scand. J. Lab. Invest. PubMed Scopus Google Scholar, M.S. Hasty K.A. C.L. PubMed Scopus Google Scholar). The for all of these was by the of the of the media for Western the cells were with phosphate-buffered and for with 199 analysis was from cultured RSF, fibroblasts, fibroblasts, HUVEC, and In addition, was isolated from human bone and and from PMN in after and were as described N. Biochem. 1987; PubMed Scopus Google Scholar). analysis was as described J. T. Scholar). reverse transcription of was reverse transcribed as described R. L. Biochem. J. PubMed Scopus Google Scholar), using as a at for 30 at for 30 and at for 30 were using a for MMP-8 MMP-1 or other were on the (9Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google MMP-8 and MMP-8 The size of the was M.S. K. M. J. PubMed Scopus Google Scholar). for the expressed gene were as described A. A. K. Biochem. 1991; PubMed Scopus Google size analysis was also on human J. T. Scholar). The from human bone peripheral blood PMN, and human fibroblasts were and J. T. Scholar). The for MMP-8 was obtained by of mRNA from bone marrow and was by Western cell culture or PMN MMP-8 was and in of on and to was by with supplemented with for at The were either with human MMP-8 J. Vissers M.C.M. Winternbourn C.C. Biochem. J. 1990; 270: 809-814Crossref PubMed Scopus (71) Google Scholar, U. J. R. Knäuper V. R. H. J. Chem. Biochem. 1989; Scholar) or with for at 20 the were with for at 20 the were for 30 with the avidin-biotin-horseradish peroxidase and the was using MMP-8 were by a The and were used as a and a The was using L. S. H. T. Biochem. PubMed Scopus Google Scholar). The contained 20 50 bovine and 20 were in and for by for with was and after was using a MMP-8 was from human neutrophil to Sorsa et al. T. Ding Y. Salo T. Lauhio A. Teronen O. Ingman T. Ohtani H. Andoh N. Takeha S. Konttinen Y.T. Ann. N. Y. Acad. Sci. 1994; 732: 112-131Crossref PubMed Scopus (143) Google Scholar). The of the MMP-8 to that of MMP-8 not of MMP-8 from PMN and from culture media of RSF and were using a of the described by et al. K.A. Y. K. M. Birkedal-Hansen H. 1990; PubMed Scopus Google Scholar). endoglycosidase was in 20 50 and at the with N-glycosidase was in and In the of both PMN MMP-8 or culture media of RSF and HUVEC, of endoglycosidase or of N-glycosidase was the of were by at for in the of The were after by of and for to neutrophil type MMP-8 collagenase used for was the as that used for J. Vissers M.C.M. Winternbourn C.C. Biochem. J. 1990; 270: 809-814Crossref PubMed Scopus (71) Google Scholar, U. J. R. Knäuper V. R. H. J. Chem. Biochem. 1989; Scholar). of synovial from rheumatoid arthritis patients were on and in at for and in PBS, at before the inhibition of peroxidase by the in in for 20 The were in a and the were with MMP-8, in and for in with bovine for 30 and avidin-biotin-horseradish peroxidase in for 30 the were for in a of of and were at and were in were further with and with before in in and of and of were as RSF were for with were with APMA, and for at with μm native type I or II collagen in 50 in the or of 50 μm The collagen degradation were by The of type I or type II collagen by MMP-8) was analyzed by the on at using an activity is expressed as a of collagen (5Konttinen Y.T. Lindy O. Suomalainen K. Ritchlin C. Saari H. Vauhkonen M. Lauhio A. Santavirta S. Sorsa T. Matrix. 1991; 11: 395-403Crossref PubMed Scopus (30) Google Scholar, K. Sorsa T. L.M. N. Saari H. Konttinen Y.T. 1992; PubMed Scopus Google Scholar). a we analyzed the expression of MMP-8 mRNA be detected in other than the bone a MMP-8 from was detected in peripheral blood cell from or rheumatoid cultured cell cells of the and HUVEC, whereas was observed in cultured human The of of the RSF mRNA was by analysis using a specific MMP-8 and the MMP-8 as those used for MMP-8 was on human in a that the of MMP-8 to the mRNA of the MMP-8 gene In addition, the were and which with the human MMP-8 (15Mainardi C.L. Pourmotabbed T.F. Hasty K.A. Am. J. Med. Sci. 1991; 302: 171-175Crossref PubMed Scopus (30) Google mRNA and protein expression in human RSF were for with TNF-α or and was MMP-8 of or RSF were transcribed by MMP-8 were by and to was used as a size analysis of the such as those described in were to and with MMP-8 MMP-8 analysis using were to and using the and mRNA expression in human endothelial cells. were for with TNF-α or and was MMP-8 of or were transcribed by MMP-8 were by and to marrow was used as a was used as a size analysis of the such as those described in were to and with MMP-8 of using specific were the as described in of MMP-8 expression in RSF and was by the cells with TNF-α or was analyzed by using by or by of RSF showed a and the of the was after the treatment of TNF-α, and after treatment also were observed after However, similar to those obtained for RSF The of was in or cells The of MMP-8 mRNA was also by analysis and The MMP-8 mRNA was observed in and cells, whereas using this MMP-8 mRNA be detected in cells both in fibroblasts and in endothelial cells MMP-8 is expressed and regulated by inflammatory or phorbol MMP-8 protein expression and Western analysis was on culture of was observed by using The used in this study not the fibroblast collagenase or other MMPs of these showed in Western the protein the of MMP-8 was both in and cells and was found in the culture media of RSF MMP-8 expression in RSF were by In media of RSF and from an MMP-8 of was that the most the nonglycosylated form of MMP-8 (9Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar, U. J. R. Knäuper V. R. H. J. Chem. Biochem. 1989; Scholar). of PMN MMP-8 by of neutrophil MMP-8 or endoglycosidase on Western of about and kDa in size whereas the treatment of media from or RSF not in the of secreted 50 kDa MMP-8 of MMP-8 by showed MMP-8 expression in mononuclear fibroblast-like cells in the human rheumatoid synovial whereas was observed using in vivo were found in from and not activity in culture media of RSF, with TNF-α, both native type I and II collagens the cleavage and Previous studies K. Sorsa T. L.M. N. Saari H. Konttinen Y.T. 1992; PubMed Scopus Google Scholar) have shown that doxycycline is a of MMP-8 activity with MMP-1 activity of 30 and of culture media from RSF with 50 μm doxycycline completely the specific degradation of type II collagen but had on the degradation of type I collagen RSF MMP-8 as shown before (9Hasty K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar), prefers type II collagen to type I collagen as a and its activity is specifically inhibited by were found in culture media of endothelial cells investigated further doxycycline the of MMP-8 analysis of RSF, with TNF-α or in the of doxycycline, showed a in the of MMP-8 mRNA and similar was obtained using In addition, MMP-8 protein was in cells with TNF-α or in the of doxycycline and doxycycline not only the activity of MMP-8 but also the expression of the MMP-8 protein in synovial Human neutrophil collagenase (MMP-8) is regarded as a matrix that is stored in granules and cell study that in to peripheral PMN, MMP-8 is expressed in RSF and human endothelial cells. was demonstrated by the mRNA size of Western and specific inhibition of enzyme activity by MMP-8 which is regulated in these cells by the inflammatory TNF-α, the protein is MMP-8, collagenase and collagenase-3 (MMP-13) are the that degrade types of native collagen. MMP-8 has the activity cartilage collagen type In MMP-8 is regarded as a role at of matrix MMP-8 activity was shown in R. Konttinen Y.T. Ding Y. M. Sorsa T. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar), in which the of activity to the C. S. K. J. Am. J. Respir. Med. 1994; PubMed Scopus Google Scholar), and rheumatoid arthritis H. N. K. Knäuper V. Y. T. Acta. 1996; PubMed Scopus Google Scholar). In addition, MMP-8 is thought to be the only collagenase that in cartilage degradation K. J. Biochem. J. 1994; PubMed Scopus Google Scholar). its synthesis and were to be to PMN MMP-8 activity at of tissue was thought to be by the degranulation of PMN (4Weiss S.J. N. Engl. J. Med. 1989; 320: 365-376Crossref PubMed Scopus (3847) Google Scholar, K.A. Jeffrey J.J. Hibbs M.S. Welgus H.G. J. Biol. Chem. 1987; 262: 10048-10052Abstract Full Text PDF PubMed Google Scholar, C.L. Pourmotabbed T.F. Hasty K.A. Am. J. Med. Sci. 1991; 302: 171-175Crossref PubMed Scopus (30) Google Scholar). In to we that the regulation of tissue by MMP-8 is most more and MMP-8 expression various cell types and the of we that as with other of the the expression of MMP-8 is regulated by inflammatory such as TNF-α and (19Unemori E.N. Hibbs M.S. J. Invest. 1991; PubMed Scopus Google Scholar, R. L. Biochem. J. PubMed Scopus Google Scholar). Cole and Kuettner (16Cole A.A. Kuettner K.E. Acta Orthop. Scand. 1995; 26: 98-102Crossref Scopus (39) Google Scholar) reported the expression of MMP-8 mRNA in PMN. In addition, the of MMP-8 expression in cartilage, in in human articular (16Cole A.A. Kuettner K.E. Acta Orthop. Scand. 1995; 26: 98-102Crossref Scopus (39) Google Scholar, S. Huch K. Mikecz K. Cs-Szabo G. Hasty K.A. Kuettner K.E. Cole A.A. Lab. Invest. 1996; 74: 232-240PubMed Google Scholar, 18Cole A.A. Chubinskaya S. Schumacher B. Huch K. Cs-Szabo G. Yao J. Mikecz K. Hasty K.A. Kuettner K.E. J. Biol. Chem. 1996; 271: 11023-11026Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), is of and that MMP-8 are also in various types of cells, in those from such as found in rheumatoid arthritis and The of the MMP-8 protein and 50 kDa, as as 20 kDa have been reported by T. Ding Y. Salo T. Lauhio A. Teronen O. Ingman T. Ohtani H. Andoh N. Takeha S. Konttinen Y.T. Ann. N. Y. Acad. Sci. 1994; 732: 112-131Crossref PubMed Scopus (143) Google Scholar, 12Seltzer J.L. Adams S.A. Grant G.A. Eisen A.Z. J. Biol. Chem. 1981; 256: 4662-4668Abstract Full Text PDF PubMed Google Scholar, K.A. Hibbs M.S. C. J. Biol. Chem. 1986; 261: Full Text PDF PubMed Google Scholar). The a of MMP-8 and the enzyme is found in a or an In addition, of MMP-8 protein have been detected as In this study we observed that MMP-8 was as a kDa protein in PMN. However, only the form was in RSF as well as in of the MMP-8 from PMN, the enzyme as size of which to the size of MMP-8 expressed by of the MMP-8 from RSF or showed that this secreted form of MMP-8 is or at all with the PMN be that stored MMP-8 in PMN whereas a or nonglycosylated form is secreted as a similar to other secreted a used MMP-8 activity at but not that of MMP-1 T. Ding Y. Salo T. Lauhio A. Teronen O. Ingman T. Ohtani H. Andoh N. Takeha S. Konttinen Y.T. Ann. N. Y. Acad. Sci. 1994; 732: 112-131Crossref PubMed Scopus (143) Google Scholar, K. Sorsa T. L.M. N. Saari H. Konttinen Y.T. 1992; PubMed Scopus Google Scholar, A. Salo T. Ding Y. Konttinen Y.T. H. J. L.M. Sorsa T. 1994; PubMed Scopus Google Scholar). these studies that the activity of MMP-8, the nonglycosylated form secreted by RSF and HUVEC, is inhibited by 50 μm Lauhio et al. A. Salo T. Ding Y. Konttinen Y.T. H. J. L.M. Sorsa T. 1994; PubMed Scopus Google Scholar) have shown that doxycycline treatment MMP-8 in arthritis. The of this is not we investigated doxycycline affect MMP-8 It was observed that after with 50 μm doxycycline in RSF as well as in both MMP-8 mRNA and MMP-8 protein expression was that doxycycline not only MMP-8 activity but also its respect to the demonstrated effects of doxycycline T. Ding Y. Salo T. Lauhio A. Teronen O. Ingman T. Ohtani H. Andoh N. Takeha S. Konttinen Y.T. Ann. N. Y. Acad. Sci. 1994; 732: 112-131Crossref PubMed Scopus (143) Google Scholar, A. Salo T. Ding Y. Konttinen Y.T. H. J. L.M. Sorsa T. 1994; PubMed Scopus Google Scholar, A. Salo T. L. J. L.M. Sorsa T. 1995; PubMed Google Scholar) and the modified L.M. Suomalainen K. Sorsa T. 1992; Google Scholar), are to the recent that MMP-8 exerts (17Chubinskaya S. Huch K. Mikecz K. Cs-Szabo G. Hasty K.A. Kuettner K.E. Cole A.A. Lab. Invest. 1996; 74: 232-240PubMed Google Scholar, K. J. Biochem. J. 1994; PubMed Scopus Google Scholar). the of the and in vivo and in T. Ding Y. Salo T. Lauhio A. Teronen O. Ingman T. Ohtani H. Andoh N. Takeha S. Konttinen Y.T. Ann. N. Y. Acad. Sci. 1994; 732: 112-131Crossref PubMed Scopus (143) Google Scholar, A. Salo T. Ding Y. Konttinen Y.T. H. J. L.M. Sorsa T. 1994; PubMed Scopus Google Scholar, A. Salo T. L. J. L.M. Sorsa T. 1995; PubMed Google Scholar), this inhibition of degrading activity be by doxycycline in vivo A. Salo T. L. J. L.M. Sorsa T. 1995; PubMed Google Scholar), at the of both proteolytic activity and has that human synovial fibroblasts, endothelial cells and A.A. Chubinskaya S. Schumacher B. Huch K. Cs-Szabo G. Yao J. Mikecz K. Hasty K.A. Kuettner K.E. J. Biol. Chem. 1996; 271: 11023-11026Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) MMP-8 doxycycline and also modified be for the degradation of both articular collagen and in In MMP-8 was to be expressed by neutrophils during their in bone and MMP-8 was to as neutrophil The of the study that in to peripheral blood several cells, such as fibroblasts and endothelial cells, are able to MMP-8 mRNA and protein both in vivo and in we that in human neutrophil collagenase (MMP-8) be to as and for are to for the of synovial