36
Agents and Actions vol. 8/1-2 (1978)
Birkh~iuser Verlag, Basel
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis by EDWARD D . HARRIS, JR., CAROL A. VATER, CARLO L. MA1NARDI Dartmouth Medical School, Hanover, New Hampshire, USA and ZENA WERB
University of California, San Francisco, Cal. USA
Abstract Remodelling of connective tissue and its destruction in rheumatoid arthritis is related to collagenolysis. Study of collagenase released by rheumatoid synovial cells has indicated that the enzyme is released in latent form from adherent synovial cells in culture. As a latent enzyme it is protected from eomplexing with u 2 maeroglobulin, the principal protelnase inhibitor. Activation in vlvo is very likely caused by proteases which destroy or complex with a portion of eollagenase responsible for its latency. Recent data suggest that the latent eollagenase is an enzymeinhibitor complex and not a true zymogen.
Introduction It is appropriate that there are m a n y different approaches to the study of rheumatoid arthritis because the full-blown lesion in this disease is generated by the contribution and interaction of multiple humoral and cellular systems. Figure 1 demonstrates the end stage of severe disease, something we must work on and study aggressively in hopes of avoiding. The principal structural component of bone, cartilage, tendons and ligaments is collagen. Our interests have been somewhat narrowly focussed upon the w a y in which collagen is degraded in RA, but we must emphasize that this is not because we view collagenase as the sun around which all other mediators o f joint destruction revolve, but that it is something we know how to study! Despite our narrow focus, as we delve more and more into the cellular reality of rheumatoid synovitis, the inextricable interrelationship of these m a n y components becomes more and more obvious. For example, the studies o f DAYER and KRANE [ 1] have given us evidence that products of activated lymphocytes and macrophages stimulate release of collagenolytic enzymes from monolayer cultures of heterogeneous mixtures o f
Figure 1 Severe erosive, destructive rheumatoid arthritis. The patient, 62-year-old female, had progressive destructive disease despite maximal anti-inflammatory drug therapy. In 1973
silastic joint replacement of the second, third, fourth and fifth metacarpophalangeal joints (MCPJ) of the right hand was carried out by Dr Forest Brown. The translucent protheses are visible, particularly in the second and fifth joints. Much function and good alignment was restored. This roentgenogram was taken in 1974, a few days before the left MCPJ were replaced. In addition to MCPJ damage, the bones were markedly osteopenic. The left wrist had a great deal of destruction with severe erosions of radius and ulnar heads. synovial cells. And as will be seen, many components of plasma m a y affect activity of eollagenolytic enzymes. A note of philosophy of biological regulation m a y be in order here. Collagen is a protein which forms readily into insoluble aggregates which have a relatively long biologic half-life. Whatever their fate, it is a prolonged one in comparison with most biological events. In addition, the
37
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
collagen fibrils in extracellular tissue are insoluble, and the first steps in degradation probably are designed to take place extracellularly at the pH and ionic strength found there. The cell, in a sense, has lost control of degradation once the enzymes which it releases have been extruded from the cells. It is only logical to presume, therefore, that initial events in collagen breakdown are part of a carefully regulated system.
Cellular regulation of collagen breakdown latent eollagenase In Figure 3 is shown a hypothesis formed from the data we have collected on what happens to rheumatoid synovial collagenase after its release in latent form from synovial cells. As a latent enzyme, collagenase is protected from being complexed by the principal proteinase inhibitor of serum, tt2 macroglobulin (azM). It has been shown that latent enzyme can be activated in the presence of cr2M if sufficient activating proteinase (trypsin or plasmin) is added to Mechanisms for collagenolysis Figure 2 shows a summary of known saturate cr2M as well as activate latent enzyme mechanisms for collagenolysis. Large aggregates [4]. Figure 4 shows results of elution of two of collagen in fibril form are cleaved either by samples from a column of agarose. One (O----O) collagenase acting directly upon individual was collagenase activated by trypsin with subsemolecules or by depolymerizing enzymes, such as quent inhibition of "trypsin by soybean trypsin the polymorphonuclear leukocyte enzymes inhibitor and then addition of cr2M. Almost no elastase or cathepsin G [2]. These, acting at active collagenase activity was eluted from this neutral pH of the extracellular space, have column and yet, after treatment of the peak specificity for non-helical, amino-terminal regions containing a2M with NaSCN, a chaotropic agent of collagen and can thereby solubilize single (Fig. 5), some collagenase was found. This monomers of collagen which can undergo ther- indicated that the active collagenase complexed mal denaturation to gelatin or first be cleaved by with tt2M, eluted with it from the column, and yet collagenase. Both insoluble as well as soluble could be freed and demonstrated active if the t~2M forms of collagen may be phagocytosed by cells itself was destroyed. Latent enzyme mixed with in the rheumatoid pannus and degraded intra- cr2M and then activated with sufficient trypsin cellularly by lysosomal enzymes such as cathep- to complex all the o~2M as well as activate sin B or Etherington's collagenolytic cathepsin collagenase (0--------0) passed through the [3]. Extracellularly, neutral proteinases and the column separate from a2M (Fig. 4) and then less well characterized peptidases may degrade could be shown to degrade collagen effectively. products of primary collagenolysis even further. We do not yet know whether or not the fl~ antiPATHWAYS 'IN VIVO' FOR COLLAGEN DEGRADATION INTRACELLULAR PHAGOLYSOSOMES pH < 6.0
EXTRACELLULARTISSUES pH 6.6- 7.6 COLLA6ENASE
Moleculor weight
I nc~nh]hl~
nnnr~,onfm~
COLLAGEN FIBER AND FIBRILS~
.___~ I PMN ELASTASE B "l r G Cathepsin Collogenolyti( I COLLAGENASECOLLAGENMONOMER cathepsin Fe0~i~st
300,000
~~~i~i ;i ~i ~i:~:lHERMAU//~DENITURATIONii:~ii ~: 100,000
GELATIN POLYPEPTIDES
I
NEUTRALPROI'EINASE S Pz Peptidoses Di ond Tri Peptidoses
ipemidoses Lysosomal
75,000 "A" 25,000 "B" I O, 000
5,000 < 1,000
EXCRETION IN URINEOR COMPLETECATABOLISM
Figure 2
This scheme of potential in vivo pathways of collagen degradation is explained in the text [from L. SOLKOLOFF, Ed., The Joints and Synovial Fluid, Academic Press, New York (in press with permission).
38
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
ACTIVATION OF
ACTIVATION
COLLAGENASE A N D CLEAVAGE
BINDING
OF PLASMINOGEN
OF COLLAGEN INHIBITION
SECRETION
TO COLLAGEN
AND/OR
Figure 3 A scheme detailing the sequence from release of latent eollagenase by cells to activation and the initiation of coliagenolysis. EL = latent collagenase; E' = active collagenase; P A = plasminogen activator; N - - C = one collagen molecule; ct2M = a 2 macroglobulin; P = plasmin; P l g = plasminogen.
o--o t.
enzyme has a capacity to bind to fibrillar substrate. Binding is probably facilitated by the presence of serum proteins, particularly tr2M and gamma globulin.
[(EL+ T)+SBTI] + (/2 M - [(EL+ O~2M)+ T] +SBTI
//~
BIOGEL -
S
5
I0
15
~
o
20
25
30
35
40
FRACTIONS
Figure 4
Elution from Bio Gel A-1.5 of collagenase activated before (O----O) or after (0------0) mixture with c~2M.The peak of ohM was recognizedby immunodiffusionin agar of column eluates against antibody to human ~M. One unit of colla genase equals 1 #g collagen fibrils degraded/min at 37~ The solid line represents absorption at 280 nm. coUagenase -- a quite specific inhibitor [5] - is capable of complexing with latent collagenase. Despite its lack of catalytic activity, the latent
T h e l a s t step - a c t i v a t i o n
Then, presumably, the system awaits activation. We do not yet know the half-life of enzyme bound to substrate under conditions found in vivo, but we have demonstrated that latent enzyme bound to fibrils can be activated by proteinases. We have accumulated compelling circumstantial evidence that plasmin may be involved in this activation in vivo [4]. One reason is that on a molar basis plasmin is a better activator of latent collagenase than trypsin, the enzyme most often successfully used to activate latent collagenases. The second is that heterogeneous rheumatoid synovial cell cultures produce a plasminogen activator. Using data accumulated in this system (Fig. 6) we have shown that coUagenolysis in this reconstituted system can be initiated simply by adding plasminogen to the cultures of synovial cells on collagen fibrils. The evidence for presence of a plasminogen activator in cultures of rheumatoid synovium was
39
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
treated dog serum (to inactivate t;t2M ) and in acid-treated, plasminogen-depleted dog serum to which plasminogen had been added. Serum-free cultures, as well, produced plasminogen activator [4]. Plasmin, therefore, may be an important enzyme for collagenolysis even though it has no action on collagen itself. It has been appreciated in recent years that plasmin can activate C1 to C1 esterase and C3 to C3a and C3b in the complement system as well as having fibrinolytic capabilities. Serving as an activator of latent collagenase may be a primary function of plasmin. Although proof is lacking, the hypothesis is an appealing one that plasminogen activator and latent collagenase may be secreted by cells involved in neovascularization of tissue. After activation by plasminogen activator, plasmin could activate latent collagenase bound to fibrils and the synergism of plasmin and collagenase could facilitate migration and growth of capillaries into tissue during remodelling and/or destruction of connective tissue. Figure 5 SDS acrylamide gels of collagen in solution incubated at 35~ with various mixtures. The y, fl and ct chains of collagen run above the 3/4 length degradation products flA and a A produced by specific collagenolysis, a B, the 1/4 length fragment, runs at the buffer front in these 5% gels. E L = latent collagenase; tt2M = a,2 macroglobulin; T = trypsin; SBTI = soybean trypsin inhibitor; NaSCN = sodium thioeyanate; E' = trypsin activated collagenase.
gathered by plating out cells for culture o n 1125fibrin in culture dishes. Fibrinolysis was measured by counting soluble non-sedimentable radioactivity in the medium. Fibrinolysis was found in cultures of cells incubated with acid-
labelled
The nature o f latent collagenase - zymogen vs. enzyme-inhibitor complex
What form of molecule is iatent collagenase? The question is important to answer, for activation of a true zymogen by proteolytic cleavage is an irreversible process, whereas dissociation of an enzyme-inhibitor complex may be reversible. Our current studies are directed toward solving this problem (Harris, Mainardi, and Vater, submitted for publication). From the lead of SELLERS et al. [6], we have used successfully organic mercurial corn-
~RADIOACTIVE COLLAGEN SOLUTION C
FILMof RADIOACTIVE / COLLAGENFIBRILS
~
MOIST, ~ THEN D R Y H E A ~
~
__ ~
I~//J////////////////////////~,~
J (~)~ASH..~ ' ~ ' - j b -
CELLSUPON RADIOACTIVE COLLAGENFIBRILS
~
EXTENSIVELY
|
Figure 6
CULTURE CELLS --with or without serum --with orwithout plasminogen
P.A.
COUNTALIQUOTPORTIONTO ' MEASUREACTIVATIONCYCLE:
EL
""PLASM,:-';>"
A reconstituted system to demonstrate activation of E L released by synovial cells cultured upon dry collagen film. P.A. = plasminogen activator; E L = latent eollagenase; E' = active collagenase.
40 pounds to activate collagenase. Mersalyl, a compound which has been used as a diuretic, is one of these. In the transition from inactive percursor to active enzyme, mersalyl effects a drop in molecular weight from ~47,000 daltons to 33,000 daltons. In addition, in serum-free culture medium containing almost entirely latent eollagenase an excess of a collagenase inhibitor has been found which, when added to activated enzyme, inhibits it. Although the source of this inhibition is unknown, the implications of the study are dear; rheumatoid synovial collagenase is probably not released from cells as a true zymogen, but as enzyme complex with an inhibitor. The inhibitor effdctively protects the enzyme from complexing with serum inhibitors.
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
Rheumatoid Synoviol Cells
3O 't-I
0.
"~ 20
~ C
uJ
I0
Therapeutic goals The cells that one finds in dissociated, adherent rheumatoid synovial cell cultures are often dominated by a strange cell type: the socalled 'dendritic cell' (Fig. 7). These stellate cells appear to be present in higher concentration in cultures producing more collagenase. W h a t is their origin? This is unknown. Perhaps they are a peculiar marrow-derived cell which have ' h o m e d ' to the inflamed joint. Perhaps a membrane-active agent in the inflammatory process has distorted a synovial cell sufficiently to make it produce large quantities of proteolytic e n z y m e . . , the answer is unknown. One advantage of the cell culture system is
Figure 7
'Dendritic cells' from rheumatoid synovium adherent to coverslips in tissue culture. These cells were dissociated from small pieces of synovium by bacterial collagenase and trypsin. They were washed and then plated out in low density in presence of serum. The percentage of this type of dendritic cell in an adherent system varies from 5% to 80%. Amounts of collagenase produced is proportional to the percentage of dendritic cells, x 200.
--I --I
8
b
,o-" ,o '~ ,o' ,o' ,o' ,o' PREDNISOLONE (moles/liter)
Figure 8
Inhibition by prednisolone of release of latent collagenase by adherent rheumatoid synovial cells in culture. With dexamethasone the curve shifts to the left, consistent with the greater potency of this drug. Synovium was dissociated, cells were grown on coverslips until confluence, and, in the absence of serum, prednisolone at various concentrations was added.
the potential for study of therapeutic alternatives in vitro. It seems not so important or practical to inhibit action of coUagenase. It does seem worth while finding ways to specifically inhibit production of collagenase. A number of investigators have shown already that corticosteroids in very small doses inhibit release of coUagenase (latent and/or active) from cells in culture. Mainardi (unpublished results) has demonstrated that concentrations of prednisolone which can be achieved easily in vivo, inhibit collagenase release from rheumatoid synovial cultures (Fig. 8). 10 -s M prednisolone is a very low concentration, and could possibly be achieved in vivo by doses of drug which produce no supression o f the hypothalamo-pituitary-adrenal axis. Our system of cells grown on collagen may provide a model for the assessment of the therapeutic potential of drugs aimed at the proliferative lesion in R A .
Acknowledgments
This work was supported by NIH grant AM14780 and by grants from the National as well as New Hampshire Chapters of The Arthritis Foundation.
41
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
References [1] J.-M. DAYER, R.G.G. RUSSELL and S.M. KRANE, Collagenase Production by Rheumatoid Synovial Cells: Stimulation by a Human Lymphocyte Factor, Science 195, 181-183 (1977). [2] P.M. STARKEY,A.J. BARRETTand M.C. BURLEIGH,The Degradation of Articular Collagen by Neutrophil Proteinases, Biochim. Biophys. Acta 483, 386-397 (1977). [3] D.J. ETHEmNGTON, The Nature of the Collagenolytic Cathepsin of Rat Liver and its Distribution in Other Rat Tissues, Biochem. J. 127, 685-692 (1972); and M.C. BURLEIGH,A.J. BARRETTand G.S. LAZARUS,Cathepsin B j a Lysosomal Enzyme that Degrades Native Collagen, Biochem J. 137, 387-398 (1974).
[4] Z. WERB, C.L. MAINARDI, C.A. MATER and E.D. HARRIS, JR., Endogenous Activation of Latent Collagenase by Rheumatoid Synovial Cells. Evidence for a Role of Plasminogen Activator, New Engl. J. Med. 296, 1017-1023 (1977). [5] D.E. WOOLLEY, D.R. ROBERT and J.M. EVANSON, Small Molecular Weight fl~ Serum Protein which Specifically Inhibits Human Collagenases, Nature (Lond.) 261, 325-327 (1976). [6] A. SELLERS, E. CARTWRIGHT, G. MURPHY and J.J. REYNOLDS, Evidence that Latent Collagenases are Enzyme-Inhibitor-Complexes, Biochem. J. 163, 303307 (1977).
DISCUSSION
Question If you establish adherent cultures from nonrheumatoid tissue, do you get these dendritic cells, and do you get latent collagenase? E.D. Harris, Jr., USA I wish it were easier to get a lot of cells from non-rheumatoid tissue. It is extremely difficult to prepare cultures from normal synovia, and frankly, we have been unable to get non-inflamed, non-proliferative synovia to examine sufficiently in this respect. This dendritic cell to us is extremely fascinating. We don't know where it comes from. It could be a macrophage, it could be an altered synovial cell, it could have yet another origin, but it is an exciting cell functionally. Question If you establish cell cultures by the explant technique, will rheumatoid synovial cell secrete latent collagenase? E.D. Harris, Jr., USA As Dr Bill Castor showed some years ago, fibroblast-like cells grow out of synovial explants after some time. Cells from rheumatoid synovium are both qualitatively and quantitatively somewhat different from cells from nonrheumatoid synovium. They secrete more hyaluronate of a lower molecular weight, they produce more lactate, but we were unable to find significant secretion of collagenase in these cultures. H. Berger, USA May I refer to my own work? We found that normal synovial cells, that means cells from patients with non-rheumatoid disease, do secrete plasminogen activator but do not secrete collagenase or fl-glucuronidase. Cell cultures estab-
lished from synovium of patients with rheumatoid arthritis or systemic lupus do not secrete either plasminogen activator or collagenase or any other enzymes, and we are suggesting that the normal function of synovium is to secrete plasminogen activator which helps to clear any fibrin deposition and that the absence of plasminogen activator secretion in rheumatoid arthritis would perpetuate inflammatory response. Inflammation calls in macrophages which clear the fibrin but will also cause tissue destruction by releasing collagenase and elastase.
E.D. Harris, Jr., USA This is a very interesting hypothesis. M. Baggiolini, Switzerland My associates Dr Wiesinger and Dr Schnyder have done similar experiments. They find that cells that grow out from explants of rheumatoid synovium do secrete plasminogen activator. Actually they secrete three to five times more than control (i.e. non-rheumatoid) synovial cells. M. Ziff, USA Is the dendritic cell peculiar to synovial cell suspension? Or can you get such cells or cells with their functional activities from cultured monocytes or macrophages? E.D. Harris, Jr., USA I haven't had very much experience with macrophages. Siamon, or Dr Allison, do you see dendritic cells in your cultures? S. Gordon, U.K. Macrophages can have a very pleomorphic appearance, but they do not look like these dendritic cells.
42
Cellular Control of Collagen Breakdownin RheumatoidArthritis
M. Ziff,, USA But do macrophages release collagenase?
E.D. Harris, Jr., USA You said it!
S. Gordon, U.K. Normal macrophages will not secrete collagenase, but activated macrophages do secrete latent coUagenase which can then be activated by plasmin.
M. Ziff, USA So this cell may well be a macrophage-like cell.
M.A. Scheinberg, Brazil We have published a paper last year in 'Arthritis and Rheumatism' showing that some of these dendritic cells have C3b and Fc receptors. This supports what Dr Ziff has just said, namely that these are probably phagocytes from the macrophage line.
Agents and Actions vol. 8/1-2 (1978)
Birkh~iuser Verlag, Basel
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis by EDWARD D . HARRIS, JR., CAROL A. VATER, CARLO L. MA1NARDI Dartmouth Medical School, Hanover, New Hampshire, USA and ZENA WERB
University of California, San Francisco, Cal. USA
Abstract Remodelling of connective tissue and its destruction in rheumatoid arthritis is related to collagenolysis. Study of collagenase released by rheumatoid synovial cells has indicated that the enzyme is released in latent form from adherent synovial cells in culture. As a latent enzyme it is protected from eomplexing with u 2 maeroglobulin, the principal protelnase inhibitor. Activation in vlvo is very likely caused by proteases which destroy or complex with a portion of eollagenase responsible for its latency. Recent data suggest that the latent eollagenase is an enzymeinhibitor complex and not a true zymogen.
Introduction It is appropriate that there are m a n y different approaches to the study of rheumatoid arthritis because the full-blown lesion in this disease is generated by the contribution and interaction of multiple humoral and cellular systems. Figure 1 demonstrates the end stage of severe disease, something we must work on and study aggressively in hopes of avoiding. The principal structural component of bone, cartilage, tendons and ligaments is collagen. Our interests have been somewhat narrowly focussed upon the w a y in which collagen is degraded in RA, but we must emphasize that this is not because we view collagenase as the sun around which all other mediators o f joint destruction revolve, but that it is something we know how to study! Despite our narrow focus, as we delve more and more into the cellular reality of rheumatoid synovitis, the inextricable interrelationship of these m a n y components becomes more and more obvious. For example, the studies o f DAYER and KRANE [ 1] have given us evidence that products of activated lymphocytes and macrophages stimulate release of collagenolytic enzymes from monolayer cultures of heterogeneous mixtures o f
Figure 1 Severe erosive, destructive rheumatoid arthritis. The patient, 62-year-old female, had progressive destructive disease despite maximal anti-inflammatory drug therapy. In 1973
silastic joint replacement of the second, third, fourth and fifth metacarpophalangeal joints (MCPJ) of the right hand was carried out by Dr Forest Brown. The translucent protheses are visible, particularly in the second and fifth joints. Much function and good alignment was restored. This roentgenogram was taken in 1974, a few days before the left MCPJ were replaced. In addition to MCPJ damage, the bones were markedly osteopenic. The left wrist had a great deal of destruction with severe erosions of radius and ulnar heads. synovial cells. And as will be seen, many components of plasma m a y affect activity of eollagenolytic enzymes. A note of philosophy of biological regulation m a y be in order here. Collagen is a protein which forms readily into insoluble aggregates which have a relatively long biologic half-life. Whatever their fate, it is a prolonged one in comparison with most biological events. In addition, the
37
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
collagen fibrils in extracellular tissue are insoluble, and the first steps in degradation probably are designed to take place extracellularly at the pH and ionic strength found there. The cell, in a sense, has lost control of degradation once the enzymes which it releases have been extruded from the cells. It is only logical to presume, therefore, that initial events in collagen breakdown are part of a carefully regulated system.
Cellular regulation of collagen breakdown latent eollagenase In Figure 3 is shown a hypothesis formed from the data we have collected on what happens to rheumatoid synovial collagenase after its release in latent form from synovial cells. As a latent enzyme, collagenase is protected from being complexed by the principal proteinase inhibitor of serum, tt2 macroglobulin (azM). It has been shown that latent enzyme can be activated in the presence of cr2M if sufficient activating proteinase (trypsin or plasmin) is added to Mechanisms for collagenolysis Figure 2 shows a summary of known saturate cr2M as well as activate latent enzyme mechanisms for collagenolysis. Large aggregates [4]. Figure 4 shows results of elution of two of collagen in fibril form are cleaved either by samples from a column of agarose. One (O----O) collagenase acting directly upon individual was collagenase activated by trypsin with subsemolecules or by depolymerizing enzymes, such as quent inhibition of "trypsin by soybean trypsin the polymorphonuclear leukocyte enzymes inhibitor and then addition of cr2M. Almost no elastase or cathepsin G [2]. These, acting at active collagenase activity was eluted from this neutral pH of the extracellular space, have column and yet, after treatment of the peak specificity for non-helical, amino-terminal regions containing a2M with NaSCN, a chaotropic agent of collagen and can thereby solubilize single (Fig. 5), some collagenase was found. This monomers of collagen which can undergo ther- indicated that the active collagenase complexed mal denaturation to gelatin or first be cleaved by with tt2M, eluted with it from the column, and yet collagenase. Both insoluble as well as soluble could be freed and demonstrated active if the t~2M forms of collagen may be phagocytosed by cells itself was destroyed. Latent enzyme mixed with in the rheumatoid pannus and degraded intra- cr2M and then activated with sufficient trypsin cellularly by lysosomal enzymes such as cathep- to complex all the o~2M as well as activate sin B or Etherington's collagenolytic cathepsin collagenase (0--------0) passed through the [3]. Extracellularly, neutral proteinases and the column separate from a2M (Fig. 4) and then less well characterized peptidases may degrade could be shown to degrade collagen effectively. products of primary collagenolysis even further. We do not yet know whether or not the fl~ antiPATHWAYS 'IN VIVO' FOR COLLAGEN DEGRADATION INTRACELLULAR PHAGOLYSOSOMES pH < 6.0
EXTRACELLULARTISSUES pH 6.6- 7.6 COLLA6ENASE
Moleculor weight
I nc~nh]hl~
nnnr~,onfm~
COLLAGEN FIBER AND FIBRILS~
.___~ I PMN ELASTASE B "l r G Cathepsin Collogenolyti( I COLLAGENASECOLLAGENMONOMER cathepsin Fe0~i~st
300,000
~~~i~i ;i ~i ~i:~:lHERMAU//~DENITURATIONii:~ii ~: 100,000
GELATIN POLYPEPTIDES
I
NEUTRALPROI'EINASE S Pz Peptidoses Di ond Tri Peptidoses
ipemidoses Lysosomal
75,000 "A" 25,000 "B" I O, 000
5,000 < 1,000
EXCRETION IN URINEOR COMPLETECATABOLISM
Figure 2
This scheme of potential in vivo pathways of collagen degradation is explained in the text [from L. SOLKOLOFF, Ed., The Joints and Synovial Fluid, Academic Press, New York (in press with permission).
38
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
ACTIVATION OF
ACTIVATION
COLLAGENASE A N D CLEAVAGE
BINDING
OF PLASMINOGEN
OF COLLAGEN INHIBITION
SECRETION
TO COLLAGEN
AND/OR
Figure 3 A scheme detailing the sequence from release of latent eollagenase by cells to activation and the initiation of coliagenolysis. EL = latent collagenase; E' = active collagenase; P A = plasminogen activator; N - - C = one collagen molecule; ct2M = a 2 macroglobulin; P = plasmin; P l g = plasminogen.
o--o t.
enzyme has a capacity to bind to fibrillar substrate. Binding is probably facilitated by the presence of serum proteins, particularly tr2M and gamma globulin.
[(EL+ T)+SBTI] + (/2 M - [(EL+ O~2M)+ T] +SBTI
//~
BIOGEL -
S
5
I0
15
~
o
20
25
30
35
40
FRACTIONS
Figure 4
Elution from Bio Gel A-1.5 of collagenase activated before (O----O) or after (0------0) mixture with c~2M.The peak of ohM was recognizedby immunodiffusionin agar of column eluates against antibody to human ~M. One unit of colla genase equals 1 #g collagen fibrils degraded/min at 37~ The solid line represents absorption at 280 nm. coUagenase -- a quite specific inhibitor [5] - is capable of complexing with latent collagenase. Despite its lack of catalytic activity, the latent
T h e l a s t step - a c t i v a t i o n
Then, presumably, the system awaits activation. We do not yet know the half-life of enzyme bound to substrate under conditions found in vivo, but we have demonstrated that latent enzyme bound to fibrils can be activated by proteinases. We have accumulated compelling circumstantial evidence that plasmin may be involved in this activation in vivo [4]. One reason is that on a molar basis plasmin is a better activator of latent collagenase than trypsin, the enzyme most often successfully used to activate latent collagenases. The second is that heterogeneous rheumatoid synovial cell cultures produce a plasminogen activator. Using data accumulated in this system (Fig. 6) we have shown that coUagenolysis in this reconstituted system can be initiated simply by adding plasminogen to the cultures of synovial cells on collagen fibrils. The evidence for presence of a plasminogen activator in cultures of rheumatoid synovium was
39
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
treated dog serum (to inactivate t;t2M ) and in acid-treated, plasminogen-depleted dog serum to which plasminogen had been added. Serum-free cultures, as well, produced plasminogen activator [4]. Plasmin, therefore, may be an important enzyme for collagenolysis even though it has no action on collagen itself. It has been appreciated in recent years that plasmin can activate C1 to C1 esterase and C3 to C3a and C3b in the complement system as well as having fibrinolytic capabilities. Serving as an activator of latent collagenase may be a primary function of plasmin. Although proof is lacking, the hypothesis is an appealing one that plasminogen activator and latent collagenase may be secreted by cells involved in neovascularization of tissue. After activation by plasminogen activator, plasmin could activate latent collagenase bound to fibrils and the synergism of plasmin and collagenase could facilitate migration and growth of capillaries into tissue during remodelling and/or destruction of connective tissue. Figure 5 SDS acrylamide gels of collagen in solution incubated at 35~ with various mixtures. The y, fl and ct chains of collagen run above the 3/4 length degradation products flA and a A produced by specific collagenolysis, a B, the 1/4 length fragment, runs at the buffer front in these 5% gels. E L = latent collagenase; tt2M = a,2 macroglobulin; T = trypsin; SBTI = soybean trypsin inhibitor; NaSCN = sodium thioeyanate; E' = trypsin activated collagenase.
gathered by plating out cells for culture o n 1125fibrin in culture dishes. Fibrinolysis was measured by counting soluble non-sedimentable radioactivity in the medium. Fibrinolysis was found in cultures of cells incubated with acid-
labelled
The nature o f latent collagenase - zymogen vs. enzyme-inhibitor complex
What form of molecule is iatent collagenase? The question is important to answer, for activation of a true zymogen by proteolytic cleavage is an irreversible process, whereas dissociation of an enzyme-inhibitor complex may be reversible. Our current studies are directed toward solving this problem (Harris, Mainardi, and Vater, submitted for publication). From the lead of SELLERS et al. [6], we have used successfully organic mercurial corn-
~RADIOACTIVE COLLAGEN SOLUTION C
FILMof RADIOACTIVE / COLLAGENFIBRILS
~
MOIST, ~ THEN D R Y H E A ~
~
__ ~
I~//J////////////////////////~,~
J (~)~ASH..~ ' ~ ' - j b -
CELLSUPON RADIOACTIVE COLLAGENFIBRILS
~
EXTENSIVELY
|
Figure 6
CULTURE CELLS --with or without serum --with orwithout plasminogen
P.A.
COUNTALIQUOTPORTIONTO ' MEASUREACTIVATIONCYCLE:
EL
""PLASM,:-';>"
A reconstituted system to demonstrate activation of E L released by synovial cells cultured upon dry collagen film. P.A. = plasminogen activator; E L = latent eollagenase; E' = active collagenase.
40 pounds to activate collagenase. Mersalyl, a compound which has been used as a diuretic, is one of these. In the transition from inactive percursor to active enzyme, mersalyl effects a drop in molecular weight from ~47,000 daltons to 33,000 daltons. In addition, in serum-free culture medium containing almost entirely latent eollagenase an excess of a collagenase inhibitor has been found which, when added to activated enzyme, inhibits it. Although the source of this inhibition is unknown, the implications of the study are dear; rheumatoid synovial collagenase is probably not released from cells as a true zymogen, but as enzyme complex with an inhibitor. The inhibitor effdctively protects the enzyme from complexing with serum inhibitors.
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
Rheumatoid Synoviol Cells
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Therapeutic goals The cells that one finds in dissociated, adherent rheumatoid synovial cell cultures are often dominated by a strange cell type: the socalled 'dendritic cell' (Fig. 7). These stellate cells appear to be present in higher concentration in cultures producing more collagenase. W h a t is their origin? This is unknown. Perhaps they are a peculiar marrow-derived cell which have ' h o m e d ' to the inflamed joint. Perhaps a membrane-active agent in the inflammatory process has distorted a synovial cell sufficiently to make it produce large quantities of proteolytic e n z y m e . . , the answer is unknown. One advantage of the cell culture system is
Figure 7
'Dendritic cells' from rheumatoid synovium adherent to coverslips in tissue culture. These cells were dissociated from small pieces of synovium by bacterial collagenase and trypsin. They were washed and then plated out in low density in presence of serum. The percentage of this type of dendritic cell in an adherent system varies from 5% to 80%. Amounts of collagenase produced is proportional to the percentage of dendritic cells, x 200.
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,o-" ,o '~ ,o' ,o' ,o' ,o' PREDNISOLONE (moles/liter)
Figure 8
Inhibition by prednisolone of release of latent collagenase by adherent rheumatoid synovial cells in culture. With dexamethasone the curve shifts to the left, consistent with the greater potency of this drug. Synovium was dissociated, cells were grown on coverslips until confluence, and, in the absence of serum, prednisolone at various concentrations was added.
the potential for study of therapeutic alternatives in vitro. It seems not so important or practical to inhibit action of coUagenase. It does seem worth while finding ways to specifically inhibit production of collagenase. A number of investigators have shown already that corticosteroids in very small doses inhibit release of coUagenase (latent and/or active) from cells in culture. Mainardi (unpublished results) has demonstrated that concentrations of prednisolone which can be achieved easily in vivo, inhibit collagenase release from rheumatoid synovial cultures (Fig. 8). 10 -s M prednisolone is a very low concentration, and could possibly be achieved in vivo by doses of drug which produce no supression o f the hypothalamo-pituitary-adrenal axis. Our system of cells grown on collagen may provide a model for the assessment of the therapeutic potential of drugs aimed at the proliferative lesion in R A .
Acknowledgments
This work was supported by NIH grant AM14780 and by grants from the National as well as New Hampshire Chapters of The Arthritis Foundation.
41
Cellular Control of Collagen Breakdown in Rheumatoid Arthritis
References [1] J.-M. DAYER, R.G.G. RUSSELL and S.M. KRANE, Collagenase Production by Rheumatoid Synovial Cells: Stimulation by a Human Lymphocyte Factor, Science 195, 181-183 (1977). [2] P.M. STARKEY,A.J. BARRETTand M.C. BURLEIGH,The Degradation of Articular Collagen by Neutrophil Proteinases, Biochim. Biophys. Acta 483, 386-397 (1977). [3] D.J. ETHEmNGTON, The Nature of the Collagenolytic Cathepsin of Rat Liver and its Distribution in Other Rat Tissues, Biochem. J. 127, 685-692 (1972); and M.C. BURLEIGH,A.J. BARRETTand G.S. LAZARUS,Cathepsin B j a Lysosomal Enzyme that Degrades Native Collagen, Biochem J. 137, 387-398 (1974).
[4] Z. WERB, C.L. MAINARDI, C.A. MATER and E.D. HARRIS, JR., Endogenous Activation of Latent Collagenase by Rheumatoid Synovial Cells. Evidence for a Role of Plasminogen Activator, New Engl. J. Med. 296, 1017-1023 (1977). [5] D.E. WOOLLEY, D.R. ROBERT and J.M. EVANSON, Small Molecular Weight fl~ Serum Protein which Specifically Inhibits Human Collagenases, Nature (Lond.) 261, 325-327 (1976). [6] A. SELLERS, E. CARTWRIGHT, G. MURPHY and J.J. REYNOLDS, Evidence that Latent Collagenases are Enzyme-Inhibitor-Complexes, Biochem. J. 163, 303307 (1977).
DISCUSSION
Question If you establish adherent cultures from nonrheumatoid tissue, do you get these dendritic cells, and do you get latent collagenase? E.D. Harris, Jr., USA I wish it were easier to get a lot of cells from non-rheumatoid tissue. It is extremely difficult to prepare cultures from normal synovia, and frankly, we have been unable to get non-inflamed, non-proliferative synovia to examine sufficiently in this respect. This dendritic cell to us is extremely fascinating. We don't know where it comes from. It could be a macrophage, it could be an altered synovial cell, it could have yet another origin, but it is an exciting cell functionally. Question If you establish cell cultures by the explant technique, will rheumatoid synovial cell secrete latent collagenase? E.D. Harris, Jr., USA As Dr Bill Castor showed some years ago, fibroblast-like cells grow out of synovial explants after some time. Cells from rheumatoid synovium are both qualitatively and quantitatively somewhat different from cells from nonrheumatoid synovium. They secrete more hyaluronate of a lower molecular weight, they produce more lactate, but we were unable to find significant secretion of collagenase in these cultures. H. Berger, USA May I refer to my own work? We found that normal synovial cells, that means cells from patients with non-rheumatoid disease, do secrete plasminogen activator but do not secrete collagenase or fl-glucuronidase. Cell cultures estab-
lished from synovium of patients with rheumatoid arthritis or systemic lupus do not secrete either plasminogen activator or collagenase or any other enzymes, and we are suggesting that the normal function of synovium is to secrete plasminogen activator which helps to clear any fibrin deposition and that the absence of plasminogen activator secretion in rheumatoid arthritis would perpetuate inflammatory response. Inflammation calls in macrophages which clear the fibrin but will also cause tissue destruction by releasing collagenase and elastase.
E.D. Harris, Jr., USA This is a very interesting hypothesis. M. Baggiolini, Switzerland My associates Dr Wiesinger and Dr Schnyder have done similar experiments. They find that cells that grow out from explants of rheumatoid synovium do secrete plasminogen activator. Actually they secrete three to five times more than control (i.e. non-rheumatoid) synovial cells. M. Ziff, USA Is the dendritic cell peculiar to synovial cell suspension? Or can you get such cells or cells with their functional activities from cultured monocytes or macrophages? E.D. Harris, Jr., USA I haven't had very much experience with macrophages. Siamon, or Dr Allison, do you see dendritic cells in your cultures? S. Gordon, U.K. Macrophages can have a very pleomorphic appearance, but they do not look like these dendritic cells.
42
Cellular Control of Collagen Breakdownin RheumatoidArthritis
M. Ziff,, USA But do macrophages release collagenase?
E.D. Harris, Jr., USA You said it!
S. Gordon, U.K. Normal macrophages will not secrete collagenase, but activated macrophages do secrete latent coUagenase which can then be activated by plasmin.
M. Ziff, USA So this cell may well be a macrophage-like cell.
M.A. Scheinberg, Brazil We have published a paper last year in 'Arthritis and Rheumatism' showing that some of these dendritic cells have C3b and Fc receptors. This supports what Dr Ziff has just said, namely that these are probably phagocytes from the macrophage line.
