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An in vitro model for the study of collagen degradation during acute inflammation
Pergamon Press
Life Sciences, Vol. 25, pp . 1885-1892 Printed in the U.S .A .
AN IN VITRO NADEL FOR THE STUDY OF COLLAGEN DEGRADATION DURING ACUTE INFLAMMATION D.J . Etherington + , I .A . Silver
a
and R . Gibbonst
Meat Research Institute+ , Langford Bristol BS18 7DY and Departments of Pathology* and Medicine{, University of Bristol, Medical School, Bristol BS8 1TD. (Received in final form October 17, 1979) Summary Collagen sponges, labelled with fluorescein, were implanted under the back skin of sensitized rats . These elicited an acute inflammatory reaction with cellular invasion of the sponge and the development of a fibrous capsule at the periphery. After 4 days each sponge, together with the fibrous capsule, was excised and placed into tissue culture . Degradation of the collagen sponge by the invading cells was monitored from the release of soluble fluorescein peptides into the medium . The addition of foetal calf serum caused inhibition above 5% (v/v) . Inhibitors of collagenase and neutral proteinases blocked the release of fluorescein peptides . Collagenolysis was also abolished or retarded by inhibitors of lysosomal cathepsins . The anti-inflammatory drug, dexamethasone, blocked all collagenolytic activity whereas indomethacin was without effect . This ex vivo model offers the possibility for following the activity of the invading phagocytic cells and for examining the enzymatic mechanisms involved in collagenolytis using appropriate perturbation techniques . The pioneering studies of Grossand co-workers (1,2) on the use of tissue culture for the identification and purification of mammalian collagenases have now been extended to a vast number of different tissues, both normal and diseased . Several recent reviews have appeared which describe the 1roperties and functions of this group of closely related enzymes (3 - 5) . It has been observed that collagenase is produced most abundantly from explants of diseased tissue in which there is an abnormal destruction of the collagenass structures and therefore the enzyme appears to be an important factor in the aetiology and severity of these disease processes (3-7) . The estimation of enzyme release by tissue explants during in vitro culture provides, at best, an empirical assessment of events in vivo . A direct evaluation of collagenase in tissue extracts is not generally possible as the enzyme binds tightly to its natural substrate in the tissue debris (8) or is complexed by proteinase inhibitors from the plasma and lymph (9) . Mammal ian collagenase cleaves a single specific locus in the triple helix of the collagen molecule (3-7) . It has been postulated that the action of this enzyme in the extracellular space is assisted by the neutral serine-proteinases, 0024-3205/79/221885-07$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1886
Collagen Degradation During Inflammation
Vol . 25, No . 22, 1979
elastase and cathepsin G, which are secreted by granulocytes in the tissue (10) . These latter enzymes degrade the non-helical telopeptides from which the intermolecular crosslinks originate (11) . Digestion of the released collagen fragments may then be continued intracellularly by phagocytic cells through the action of cathepsin H and cathepsin N (previously called collagenolytic cathepsin), which can also digest away the telopeptides . It is thus generally believed that there is a two-stage process for the degradation of collagen fibres in the extracellular structures (3,7,12,13) . However, it is still very unclear how effective is the initial action by the neutral proteinases in the pericellular environment of phagocytic cells . This zone may become acidic and thus block any further action by these enzymes . Preliminary studies using microelectrode probes have found that for activated macrophages the pericellular pH can fall below 5 .0 (Silver, unpublished observation) . In this laboratory a new approach has been taken in our studies of the collagen-degrading mechanisms through the development of a reproducible inflammatory model initiated by antigen-antibody complex formation against the fluorescein hapten . A preliminary report of this model has been given (14) . Materials and Methods Female Sprague-Dawley rats (8w) were sensitized with fluorescein-labelled, acid-soluble bovine collagen (15) . The antigen (1.62 mg/ml and possessing 5 .9 moles of hapten label per mole collagen in 0 .1% v/v acetic acid) was diluted 3-fold with phosphate-buffered saline (PBS) and then emulsified with an equal volume of Freund's complete adjuvant . Each animal received 0 .4 ml divided between four sub-dermal sites and the injections were repeated 2w later. Insoluble acid-washed bovine tendon collagen (16) was labelled with fluorescein (15) and sponges were prepared from this by freeze-drying a 0 .7 cm thick dispersion in 1.0% v/v acetic acid . Sponges (10 mm x 10 mm, approx . 5 mg) were sterilized with 70% v/v ethanol, immersed in PBS containing 300 units ml -1 penicillin and 300 ug ml-1 streptomycin and then implanted, in pairs, under the back skin of anaesthetized rats using sterile techniques, 2w following the second injection of antigen . After 4 days, when the acute phase of the inflammatory reaction had passed its peak, the rats were sacrificed and the sponges carefully removed together with the enclosing fibrous capsule . Each sponge was bisected once and both halves placed into 5 ml of medium 199, containing 100 units ml -1 penicillin and 100 jig ml-1 streptomycin in a 25 ml Falcon culture flask. The cultures, were maintained at 37 0 in an atmosphere of 95% air : 5% 00 2 and the medium changed every 2 days . For some experiments the medium was supplemented with foetal calf serum. Proteinase inhibitors and anti-inflammatory agents were prepared as concentrated stock solutions and then added directly to the medium as indicated. Following the fifth medium collection, each sponge residue was suspended in 10 ml of PBS containing 2 mM-2-mercaptoethanol, the collagen heat denatured and digested with 5 jig of papain at 500 for 20h . The quantity of soluble fluorescein-label in each fraction was estimated at 520 nm with a doublemonochromator fluorescence spectrophotometer using an excitation wavelength of 495 nm . Some sponges were fixed in 10% formol-saline immediately after excision or following the 10 day period in tissue culture . Histological examination was made of 4 um sections stained with haematoxylin and eosin.
Vol . 25, No . 22, 1979
Collagen Degradation During Inflammation
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Vol . 25, No . 22, 1979
SERUM IN MEDIUM 199
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FIG . 2 The release of fluorescein-peptides from explanted sponges in culture . Fluorescein-collagen sponges (approx . 5 mg) were implanted subcutaneously, in pairs, into sensitized rats (a) or into non-sensitized, control, rats of the same age (b) . Sponges were removed after 4 days and maintained at 370 in medium 199 . Foetal calf serum was incorporated to 5% and 10% (v/v) as indicated or omitted from the medium (serum-free) . The results are expressed as percentage of total recovered fluorescein-label from each sponge in the bidaily fractions (1 -5) and in the sponge residue (R) . Results When fluorescein-labelled collagen sponges were implanted into sensitized rats, an acute inflammatory reaction was elicited at the site of each sponge . The heterologous collagen fibres were rapidly degraded between the 6th and 11th day following implantation . A full description of the histopathology of this reaction will be published elsewhere (17) . Fig. la shows a photomicrograph of the fluorescein-collagen sponge when excised at 4d . Considerable fibroblast
Vol . 25, No . 22, 1979
Collagen Degradation During Inflammation
188 9
activity existed around the sponge which was totally enclosed by a granulation tissue capsule . There was a substantial inflammatory oedema with very many PMN leucocytes attaching to the collagen fibres . Many macrophages were present at this time and a few lymphocytes were to be seen also . Estimation of the solubilized fluorescein-peptides in the medium fractions and the remaining fluorescein-collagen in the residual sponge generally gave a In Fig . 2a recovery of 70-100% of the measured label in the original sponge . the maximal release of fluorescein-conjugated peptides is shown to occur in the third medium collection (days 5-6) from the serum-free medium 199 and only 16% of the label remained in the sponge after 10 days . A supplement of 5% v/v foetal calf serum caused only a small decrease in collagenolysis but higher concentrations became strongly inhibitory . The results were negative (Fig . 2b) (i .e . there was no fluorescein release) when the sponges had been implanted into non-sensitized animals, which confirmed the immunological basis of the host reaction . The cell population associated with the collagen sponge at the end of the 10 days cultivation period (Fig . lb) was seen to decrease, presumably due to migration of cells into the medium and to death of neutrophils since these are naturally short-lived and are incapable of reproduction . The addition of 5% (v/v) foetal calf serum to the medium produced little change in the histological picture . In Fig . 3 are shown the results obtained when specific proteinase inhibitors or anti-inflammatory agents were added to the culture medium . Collagenase is a metallo-proteinase (5) and requires both calcium and zinc for activation . 1,10-phenanthroline, which chelates the zinc ions, caused total inhibition withonly slight loss of cell viability (Fig . lc), Phenyl methyl sulphonyl fluoride (PMSF) will inhibit the neutral serine-proteinases (18) and this also caused total inhibition . The thiol-cathepsins, B and N, are inhibited by N-a-p-tosylL-lysine chloromethyl ketone (TLCK) (13) and addition of this compound to the medium retarded and reduced the release of collagen peptides . Chloroquin is concentrated by lysosomes (19) and at 1 mM in the medium caused total inhibition . The anti-inflammatory agent, dexamethasone, at 2 x 10 abolished all collagenolysic activity, whereas indomethacin, which in vivo specifically blocks prostaglandin synthesis (20), was totally ineffective on these explants . Discussion Fluorescein-collagen sponges are rapidly degraded in sensitized rata after about 6 days following implantation . When the implant is removed at day L, then collagenolysis can be studied under in vitro conditions using sterile tissue-culture techniques . The rate of degradation as determined from the release of soluble fluorescein peptides is closely parallel to the rate at which the sponge is resorbed in vivo . Inhibitors of collagenase, neutral serine proteinases and lysosomel thiol-cathepsins, were all shown to block collagen degradation in vitro . The differential effect of the two antiinflammatory agents, dexamethasone and indomethacin, is consistent with the observed histological changes in vivo during sponge resorption . The early invasion by PMN leucocytes causes no detectable breakdown of the implanted fluorescein-collagen fibres and indomethacin exerts its effect in vivo against this cell type . The macrophages which entered subsequently were seen to engage directly in the degradation of these fibres . The activity of this cell type is known to be readily inhibited by dexamethasone and this agent was shown to block collagenolysis completely during culture . The in vitro model we have described for the study of collagenolysis offers several potential advantages over other existing methods . Firstly, the extent
1890
Collagen Degradation During Inflammation
Vol . 25, No . 22, 1979
SERUM-FREE 199 199 " 5%FC5 1 ,10-PHENANTHROLINE (0 .5mM) 100r
0 100
0 -i100
0 100
PMSF (0 .1mM)
TLCK
(0.5mM)
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FIG. 3 The effect of proteinase inhibitors and anti-inflammatory agents on the The release of fluorescein-peptides from explanted sponges in culture . conditions are the same as in Fig . 2(a) but only serum-free medium 199 or medium supplemented with 5% (v/v) foetal calf serum (5% FCS) were tested . Each compound was prepared as a stock solution and added directly to the media to the final concentration as indicated . Results are expressed as in Fig . 2 .
Vol . 25, No . 22, 1979
Collagen Degradation During Inflammation
189 1
of collagen degradation can be accurately determined from the release of soluble fluorescein-peptides . Secondly, although the cell population varies during the course of the inflammatory reaction, it is possible to remove sponges at different times and to investigate how these changes may influence collagenolysis under the in vitro conditions . Furthermore, all stages in the resorption of collagen _in vitro can be compared with the process in vivo . Thirdly, the use of specific inhibitors can be developed to identity each enzyme in the degradation of collagen during inflammation . Fourthly, the opportunity exists to investigate the extent of phagocytic activity . Preliminary data from the electron microscope (C .A . Voyle - unpublished observation) have revealed a rapid development of collagen-containing vacuoles in the macrophage population . Lastly, the effect on collagenolysis of different agents that are known to modify cellular activity, such as anti-inflammatory drugs, can be conveniently tested using this in vitro model Acknowle dgement We are indebted to Dr Yates of the Department of Biochemistry for the use of equipment and facilities . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .
J .GROSS, and C .M . LAPIERE, Proc . Natl . Acad . Sci . U .S .A . _48, 1014-1022 . (1962) . Y . NAGAI, C .M . LAPIERE, and J . GROSS, Biochemistry 5, 3132-3140 (1966) . E .D . HARRIS, JR ., and S .M . KRANE, New Engl . J . Med . 221, 557- 563, 605- 609, 652-661 (1974) . J . GROSS, in Biochemistry of Collagen (eds . G .N . Ramachandran, and A .H . Reddi) 275-317 (Plenum, New York, 1976) . E .D . HARRIS, JR ., and E .C . CARTWRIGHT, in Proteinases in Mammalian Cells and Tissues (ed . A .J . Barrett) 249-283 (North-Holland, Amsterdam, 1977) . R . PEREZ-TAMAYO, Amer . J . Path . L32, 509-566 (1978) . D .J . ETHERINGTON, Ann . Rheum . Dis . 36, suppl . 2, 14-17 (1977) . J .F . WOESSNER, JR ., and J .N . RYAN, Bioche . Biophs . Acta . 309, 397-405 (1973) . D .E . WOOLLEY, and J .M . EVANSON, Connect . Tissue Res . ~, 31-35 (1977) . P .M . STARKEY, A .J . BARRETT, and M .C . BURLEIGH, Biochim . Biophys . Acta . 483, 386-397 (1977) . A .J . BAILEY, S .P . ROBINS, and G . BALIAN, Nature ZLI, 105-109 (1974) . M .C . BURLEIGH, in Proteinases in Mammalian Cells and Tissues (ed . A .J . Barrett) 285-309 (North-Holland, Amsterdam, 1977) . D .J . ETHERINGTON, and P .J . EVANS, Acta Biol . Med . Germ . 36, 1555-1563 (1977) . D .J .'ETHERINGTON, I .A . SILVER, and R . GIBBONS, Path . Biol . (in press) . F .S . STEVEN, A . TORRE-BLANCO, and J .A .A . HUNTER, Biochim . Biophys . Acta 4~, 188-200 (1975) . D .J . ETHERINGTON, Connect . Tissue Res ., 135 -145 (1977) . . D .J . ETHERINGTON, I .A . SILVER, and D .J . RESTALL, Br . J . Exptl . Path . (in press) . P .M . STARKEY, in Proteinases in Mammalian Cells and Tissues (ed . A .J . Barrett) 57-89 (North-Holland, Amsterdam, 1977) . M . WIBO, and B . POOLE, Cell Biol . .23, 430-440 (1974) . P .Y . SHEN, E .A . HAM, V .J . CIRILLO, and M . ZANETTI, in Prostaglandin Synthetase Inhibitors - Their Effects on Physiological Function and Pathological States (eds . H .J . Robinson, and J .R . Vane) 19-31 . (Raven, New York, 1974) .
Life Sciences, Vol. 25, pp . 1885-1892 Printed in the U.S .A .
AN IN VITRO NADEL FOR THE STUDY OF COLLAGEN DEGRADATION DURING ACUTE INFLAMMATION D.J . Etherington + , I .A . Silver
a
and R . Gibbonst
Meat Research Institute+ , Langford Bristol BS18 7DY and Departments of Pathology* and Medicine{, University of Bristol, Medical School, Bristol BS8 1TD. (Received in final form October 17, 1979) Summary Collagen sponges, labelled with fluorescein, were implanted under the back skin of sensitized rats . These elicited an acute inflammatory reaction with cellular invasion of the sponge and the development of a fibrous capsule at the periphery. After 4 days each sponge, together with the fibrous capsule, was excised and placed into tissue culture . Degradation of the collagen sponge by the invading cells was monitored from the release of soluble fluorescein peptides into the medium . The addition of foetal calf serum caused inhibition above 5% (v/v) . Inhibitors of collagenase and neutral proteinases blocked the release of fluorescein peptides . Collagenolysis was also abolished or retarded by inhibitors of lysosomal cathepsins . The anti-inflammatory drug, dexamethasone, blocked all collagenolytic activity whereas indomethacin was without effect . This ex vivo model offers the possibility for following the activity of the invading phagocytic cells and for examining the enzymatic mechanisms involved in collagenolytis using appropriate perturbation techniques . The pioneering studies of Grossand co-workers (1,2) on the use of tissue culture for the identification and purification of mammalian collagenases have now been extended to a vast number of different tissues, both normal and diseased . Several recent reviews have appeared which describe the 1roperties and functions of this group of closely related enzymes (3 - 5) . It has been observed that collagenase is produced most abundantly from explants of diseased tissue in which there is an abnormal destruction of the collagenass structures and therefore the enzyme appears to be an important factor in the aetiology and severity of these disease processes (3-7) . The estimation of enzyme release by tissue explants during in vitro culture provides, at best, an empirical assessment of events in vivo . A direct evaluation of collagenase in tissue extracts is not generally possible as the enzyme binds tightly to its natural substrate in the tissue debris (8) or is complexed by proteinase inhibitors from the plasma and lymph (9) . Mammal ian collagenase cleaves a single specific locus in the triple helix of the collagen molecule (3-7) . It has been postulated that the action of this enzyme in the extracellular space is assisted by the neutral serine-proteinases, 0024-3205/79/221885-07$02 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1886
Collagen Degradation During Inflammation
Vol . 25, No . 22, 1979
elastase and cathepsin G, which are secreted by granulocytes in the tissue (10) . These latter enzymes degrade the non-helical telopeptides from which the intermolecular crosslinks originate (11) . Digestion of the released collagen fragments may then be continued intracellularly by phagocytic cells through the action of cathepsin H and cathepsin N (previously called collagenolytic cathepsin), which can also digest away the telopeptides . It is thus generally believed that there is a two-stage process for the degradation of collagen fibres in the extracellular structures (3,7,12,13) . However, it is still very unclear how effective is the initial action by the neutral proteinases in the pericellular environment of phagocytic cells . This zone may become acidic and thus block any further action by these enzymes . Preliminary studies using microelectrode probes have found that for activated macrophages the pericellular pH can fall below 5 .0 (Silver, unpublished observation) . In this laboratory a new approach has been taken in our studies of the collagen-degrading mechanisms through the development of a reproducible inflammatory model initiated by antigen-antibody complex formation against the fluorescein hapten . A preliminary report of this model has been given (14) . Materials and Methods Female Sprague-Dawley rats (8w) were sensitized with fluorescein-labelled, acid-soluble bovine collagen (15) . The antigen (1.62 mg/ml and possessing 5 .9 moles of hapten label per mole collagen in 0 .1% v/v acetic acid) was diluted 3-fold with phosphate-buffered saline (PBS) and then emulsified with an equal volume of Freund's complete adjuvant . Each animal received 0 .4 ml divided between four sub-dermal sites and the injections were repeated 2w later. Insoluble acid-washed bovine tendon collagen (16) was labelled with fluorescein (15) and sponges were prepared from this by freeze-drying a 0 .7 cm thick dispersion in 1.0% v/v acetic acid . Sponges (10 mm x 10 mm, approx . 5 mg) were sterilized with 70% v/v ethanol, immersed in PBS containing 300 units ml -1 penicillin and 300 ug ml-1 streptomycin and then implanted, in pairs, under the back skin of anaesthetized rats using sterile techniques, 2w following the second injection of antigen . After 4 days, when the acute phase of the inflammatory reaction had passed its peak, the rats were sacrificed and the sponges carefully removed together with the enclosing fibrous capsule . Each sponge was bisected once and both halves placed into 5 ml of medium 199, containing 100 units ml -1 penicillin and 100 jig ml-1 streptomycin in a 25 ml Falcon culture flask. The cultures, were maintained at 37 0 in an atmosphere of 95% air : 5% 00 2 and the medium changed every 2 days . For some experiments the medium was supplemented with foetal calf serum. Proteinase inhibitors and anti-inflammatory agents were prepared as concentrated stock solutions and then added directly to the medium as indicated. Following the fifth medium collection, each sponge residue was suspended in 10 ml of PBS containing 2 mM-2-mercaptoethanol, the collagen heat denatured and digested with 5 jig of papain at 500 for 20h . The quantity of soluble fluorescein-label in each fraction was estimated at 520 nm with a doublemonochromator fluorescence spectrophotometer using an excitation wavelength of 495 nm . Some sponges were fixed in 10% formol-saline immediately after excision or following the 10 day period in tissue culture . Histological examination was made of 4 um sections stained with haematoxylin and eosin.
Vol . 25, No . 22, 1979
Collagen Degradation During Inflammation
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Vol . 25, No . 22, 1979
SERUM IN MEDIUM 199
0
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FIG . 2 The release of fluorescein-peptides from explanted sponges in culture . Fluorescein-collagen sponges (approx . 5 mg) were implanted subcutaneously, in pairs, into sensitized rats (a) or into non-sensitized, control, rats of the same age (b) . Sponges were removed after 4 days and maintained at 370 in medium 199 . Foetal calf serum was incorporated to 5% and 10% (v/v) as indicated or omitted from the medium (serum-free) . The results are expressed as percentage of total recovered fluorescein-label from each sponge in the bidaily fractions (1 -5) and in the sponge residue (R) . Results When fluorescein-labelled collagen sponges were implanted into sensitized rats, an acute inflammatory reaction was elicited at the site of each sponge . The heterologous collagen fibres were rapidly degraded between the 6th and 11th day following implantation . A full description of the histopathology of this reaction will be published elsewhere (17) . Fig. la shows a photomicrograph of the fluorescein-collagen sponge when excised at 4d . Considerable fibroblast
Vol . 25, No . 22, 1979
Collagen Degradation During Inflammation
188 9
activity existed around the sponge which was totally enclosed by a granulation tissue capsule . There was a substantial inflammatory oedema with very many PMN leucocytes attaching to the collagen fibres . Many macrophages were present at this time and a few lymphocytes were to be seen also . Estimation of the solubilized fluorescein-peptides in the medium fractions and the remaining fluorescein-collagen in the residual sponge generally gave a In Fig . 2a recovery of 70-100% of the measured label in the original sponge . the maximal release of fluorescein-conjugated peptides is shown to occur in the third medium collection (days 5-6) from the serum-free medium 199 and only 16% of the label remained in the sponge after 10 days . A supplement of 5% v/v foetal calf serum caused only a small decrease in collagenolysis but higher concentrations became strongly inhibitory . The results were negative (Fig . 2b) (i .e . there was no fluorescein release) when the sponges had been implanted into non-sensitized animals, which confirmed the immunological basis of the host reaction . The cell population associated with the collagen sponge at the end of the 10 days cultivation period (Fig . lb) was seen to decrease, presumably due to migration of cells into the medium and to death of neutrophils since these are naturally short-lived and are incapable of reproduction . The addition of 5% (v/v) foetal calf serum to the medium produced little change in the histological picture . In Fig . 3 are shown the results obtained when specific proteinase inhibitors or anti-inflammatory agents were added to the culture medium . Collagenase is a metallo-proteinase (5) and requires both calcium and zinc for activation . 1,10-phenanthroline, which chelates the zinc ions, caused total inhibition withonly slight loss of cell viability (Fig . lc), Phenyl methyl sulphonyl fluoride (PMSF) will inhibit the neutral serine-proteinases (18) and this also caused total inhibition . The thiol-cathepsins, B and N, are inhibited by N-a-p-tosylL-lysine chloromethyl ketone (TLCK) (13) and addition of this compound to the medium retarded and reduced the release of collagen peptides . Chloroquin is concentrated by lysosomes (19) and at 1 mM in the medium caused total inhibition . The anti-inflammatory agent, dexamethasone, at 2 x 10 abolished all collagenolysic activity, whereas indomethacin, which in vivo specifically blocks prostaglandin synthesis (20), was totally ineffective on these explants . Discussion Fluorescein-collagen sponges are rapidly degraded in sensitized rata after about 6 days following implantation . When the implant is removed at day L, then collagenolysis can be studied under in vitro conditions using sterile tissue-culture techniques . The rate of degradation as determined from the release of soluble fluorescein peptides is closely parallel to the rate at which the sponge is resorbed in vivo . Inhibitors of collagenase, neutral serine proteinases and lysosomel thiol-cathepsins, were all shown to block collagen degradation in vitro . The differential effect of the two antiinflammatory agents, dexamethasone and indomethacin, is consistent with the observed histological changes in vivo during sponge resorption . The early invasion by PMN leucocytes causes no detectable breakdown of the implanted fluorescein-collagen fibres and indomethacin exerts its effect in vivo against this cell type . The macrophages which entered subsequently were seen to engage directly in the degradation of these fibres . The activity of this cell type is known to be readily inhibited by dexamethasone and this agent was shown to block collagenolysis completely during culture . The in vitro model we have described for the study of collagenolysis offers several potential advantages over other existing methods . Firstly, the extent
1890
Collagen Degradation During Inflammation
Vol . 25, No . 22, 1979
SERUM-FREE 199 199 " 5%FC5 1 ,10-PHENANTHROLINE (0 .5mM) 100r
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DEXAMETHASONE (2x10 _8 M)
INDOMETHACIN (10-7M)
r]
-r-J `. 1 2 3 4 5 R CULTURE
____
_j
L ,
1 2 3 4 5 FRACTION
n R
FIG. 3 The effect of proteinase inhibitors and anti-inflammatory agents on the The release of fluorescein-peptides from explanted sponges in culture . conditions are the same as in Fig . 2(a) but only serum-free medium 199 or medium supplemented with 5% (v/v) foetal calf serum (5% FCS) were tested . Each compound was prepared as a stock solution and added directly to the media to the final concentration as indicated . Results are expressed as in Fig . 2 .
Vol . 25, No . 22, 1979
Collagen Degradation During Inflammation
189 1
of collagen degradation can be accurately determined from the release of soluble fluorescein-peptides . Secondly, although the cell population varies during the course of the inflammatory reaction, it is possible to remove sponges at different times and to investigate how these changes may influence collagenolysis under the in vitro conditions . Furthermore, all stages in the resorption of collagen _in vitro can be compared with the process in vivo . Thirdly, the use of specific inhibitors can be developed to identity each enzyme in the degradation of collagen during inflammation . Fourthly, the opportunity exists to investigate the extent of phagocytic activity . Preliminary data from the electron microscope (C .A . Voyle - unpublished observation) have revealed a rapid development of collagen-containing vacuoles in the macrophage population . Lastly, the effect on collagenolysis of different agents that are known to modify cellular activity, such as anti-inflammatory drugs, can be conveniently tested using this in vitro model Acknowle dgement We are indebted to Dr Yates of the Department of Biochemistry for the use of equipment and facilities . References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 .
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