Histochemical Journal 28, 229-245 (1996)
REVIEW
Phagocytosis and intracellular digestion of collagen, its role in turnover and remodelling V I N C E N T E V E R T S 1"2., E R W I N WOUTER BEERTSEN 1
V A N D E R Z E E 1, L A U R A
CREEMERS 1 and
1ExperimentaI Oral Biology Group, Department of Periodontology, Academic Centre for Dentistry Amsterdam (ACTA) and 2Laboratory of Cell Biology and Histology, Academic Medical Centre, Meibergdreef15, 1105 AZ Amsterdam, The Netherlands Received 12 September 1995 and in revised form 10 November 1995
Summary Collagens of most connective tissues are subject to continuous remodelling and turnover, a phenomenon which occurs under both physiological and pathological conditions. Degradation of these proteins involves participation of a variety of proteolytic enzymes including members of the following proteinase classes: matrix metalloproteinases (e.g. collagenase, gelatinase and stromelysin), cysteine proteinases (e.g. cathepsin B and L) and serine proteinases (e.g. plasmin and plasminogen activator). Convincing evidence is available indicating a pivotal role for matrix metalloproteinases, in particular collagenase, in the degradation of collagen under conditions of rapid remodelling, e.g. inflammation and involution of the uterus. Under steady state conditions, such as during turnover of soft connective tissues, involvement of collagenase has yet to be demonstrated. Under these circumstances collagen degradation is likely to take place particularly within the lysosomal apparatus after phagocytosis of the fibrils. We propose that this process involves the following steps: (i) recognition of the fibril by membranebound receptors (integrins?), (ii) segregation of the fibril, (iii) partial digestion of the fibril and/or its surrounding noncollagenous proteins by matrix metalloproteinases (possibly gelatinase), and finally (iv) lysosomal digestion by cysteine proteinases, such as cathepsin B and/or L. Modulation of this pathway is carried out under the influence of growth factors and cytokines, including transforming growth factor [~and interleukin lct.
Introduction The collaget~ous framework present in the extracellular environment of virtually all connective tissues undergoes degradation (Krane, 1985; Laurent, 1987; McAnulty & Laurent, 1987; Sodek, 1989). This applies not only to soft connective tissues but also to most mineralized tissues like bone and mineralized cartilage (see Delaiss6 & Vaes, 1992). The cell of pivotal importance in both synthesis and degradation of collagenous proteins in soft connective tissues is the fibroblast. Numerous in vitro studies have shown that these cells can synthesize and secrete various collagen-degrading enzymes, one of which is matrix metalloproteinase-1 (MMP-1, collagenase). This enzyme was initially described by Gross and Lapiere in 1962 and is one of the few enzymes capable of attacking the native collagen molecule. Based on this capacity, *To whom correspondence should be addressed at the Laboratory of Cell Biologyand Histology,AcademicMedicalCentre, Meibergdreef. 0018-2214 9 1996 C h a p m a n & Hall
collagenase is considered to be crucial in the degradation of collagens (e.g. Woessner, 1991). In addition to collagenase, fibroblasts produce other matrix-degrading enzymes, including matrix metalloproteinases such as gelatinase (MMP-2) or stromelysins (MMP-3 and MMP-11), serine proteinases (plasminogen activator) and cysteine proteinases (cathepsins B and L). There is convincing evidence that under a variety of conditions (morphogenesis, inflammation, metastasis) one or more of these enzymes are secreted and are active at sites undergoing digestion (see M u r p h y et al., 1991; Aznavoorian et al., 1993; Birkedal-Hansen et al., 1993; Cottam & Rees; 1993; Mignatti & Rifkin, 1993; M u r p h y & Reynolds, 1993). Far less is k n o w n about their possible involvement in physiological steady-state conditions. It is rather surprising that up until n o w no studies have appeared which demonstrate the presence of collagenase in connective tissues with a rapid turnover such as the periodontal ligament (Sodek, 1977, 1978,
230 1989; Imberman et al., 1986). Possibly the concentration in these tissues is too low to detect with currently available techniques. Degradation may occur in restricted areas in close proximity to the plasma membrane where, following its secretion, collagenase is activated by plasmin (Quigley et al., 1990). In this way, degradation of collagen or other extracellular matrix components may occur in confined areas with a minute amount of enzyme. Yet, in cases of rapid turnover one would expect to find at least some indication for the presence of collagenase. Alternatively, is degradation under these conditions effected without the involvement of this enzyme? Currently we know that under conditions characterized by normal turnover, an alternative pathway may be followed, the intracellular route (Ten Cate, 1972; Deporter & Ten Cate, 1973; Listgarten, 1973; Beertsen et al., 1974; Ten Cate & Freeman, 1974; Ten Cate & Deporter, 1975; Garant, 1976; Ten Cate et al., 1976; Beertsen & Everts, 1977). This route involves the uptake of collagen fibrils by connective tissue cells and subsequent breakdown of the fibrils in the lysosomal apparatus (Melcher &Chan, 1981; Everts & Beertsen, 1988). This review focuses on this pathway of collagen digestion, with emphasis on normal turnover and remodelling of soft connective tissues.
Intracellular collagen The presence of cross-banded collagen fibrils or fibril fragments in cytoplasmic vacuoles has been reported for almost all (soft) connective tissues. Although the most prominent cell type in which fibrillar collagen is found intracellularly is the fibroblast, the presence of collagen fibrils in cytoplasmic vacuoles has been shown in other cells, such as epithelial cells (Trelstad, 1971; Birek et al., 1980; Salonen et al., 1991), chondrocytes (Sheldon & Kimball, 1962), osteoblasts (Takahashi et al., 1986a,b; Everts et al., 1994a), macrophages (Parakkal, 1969; Deporter, 1979), smooth muscle cells (Jurukova, 1980; Jurukova & Milenkov, 1981), odontoblasts (Ishizeki et al., 1987), decidua cells (Zorn et al., 1989), cementoblasts (Yajima et al., 1989) and osteoclasts (Everts et aI., 1985a; 1988; Van Noorden & Everts, 1991). Fibroblasts
Intracellular cross-banded collagen fibrils are frequently found in fibroblasts in two types of vacuoles: those containing electron-dense material surrounding the fibrils (Fig. la) and those in which the space between the fibril and the vacuolar membrane is filled with electron-translucent material (Fig. lb; e.g. Beertsen et al., 1978; Melcher &Chan, 1981). It was suggested (Beertsen et al., 1974) that the latter type of vacuole contains fibrils recently engulfed but still in contact with the extracellular space (see also Melcher & Chan, 1981). In most
EVERTS et al. instances such vacuolar structures enclose only one or two fibrils. The electron-dense vacuoles, on the other hand, frequently contain more than one fibril and have been proven to be wholly intracellular (Melcher & Chan, 1981; Everts et al., 1985b). Several studies demonstrated acid phosphatase activity in the electron-dense type of vacuoles, indicating the presence of lysosomal enzymes (Fig. 2; Deporter & Ten Cate, 1973; Beertsen et aI., 1978; Everts et al., 1985b; Yajima, 1986, 1988). Some authors localized the enzyme also in electron-translucent vacuoles (Beertsen et al., 1978). In numerous electron-dense vacuoles, in addition to single fibrils, large and compact collagenous structures are frequently observed, suggesting that individual fibrils have aggregated side-by-side to form condensed conglomerates (Fig. 3; Dingemans & Teeling, 1994). Such broad fibrillar structures are considered to represent a next step in the digestion process. In addition to acid phosphatase activity, alkaline phosphatase also appears to be present in collagencontaining vacuoles (G6thlin & Ericsson, 1970; Deporter & Ten Cate, 1973; Ten Cate & Syrbu, 1974; Thyberg et aI., 1979). This enzyme is found almost exclusively in the electron-translucent vacuoles and is bound to the cytoplasmic membrane surrounding the collagen fibril. The functional significance of this enzyme remains to be elucidated. The presence of cross-banded collagen fibrils in the vacuolar apparatus of cells such as fibroblasts has been explained in two ways. First, the intracellular collagen may reflect an accumulation of non-excreted newlysynthesized collagen (Welsh, 1966; Trelstad, 1971; Allegra & Broderick, 1973; Renteria & Ferrans, 1976; Levine et aI., 1978; Henell et at., 1983; Birk & Trelstad, 1984; Michna, 1988; Enwemeka, 1991). Second, they may reflect phagocytosed fibrils (Ten Cate, 1972; Listgarten, 1973; Beertsen et al., 1974; Ten Cate & Deporter, 1975; Garant, 1976; Beertsen & Everts, 1977; Yajima & Rose, 1977; Rose et al., 1978; Shore & Berkovitz, 1979; Svoboda et al., 1979a,b; Rose et al., 1980; Melcher &Chan, 1981; Yamasaki et aI., 1981; Fell et al., 1986).
Synthesis or phagocytosis? .
In 1978 Bienkowsky and colleagues showed that, immediately following synthesis, a considerable amount of collagen is not secreted but is degraded intracellularly (Bienkowsky et al., 1978). It was demonstrated subsequently that this pathway of collagen digestion depends on the activity of lysosomal enzymes (Berg et al., 1984), in particular those belonging to the class of cysteine proteinases (Berg et aI., 1984; Neblock & Berg, 1984). It appeared that relatively large amounts of collagen (up to about 30% of the newly-synthesized collagen) are susceptible to lysosomal digestion in connective tissue cells (Neblock & Berg, 1984).
Phagocytosis and intracellular digestion of collagen
231
Fig. 1. Collagen-containing vacuoles in periodontal ligament fibroblasts. (a) Cross-banded collagen (arrow) surrounded by electron-dense material enclosed in a cytoplasmic vacuole. (b) Vacuole enclosing collagen fibrils (arrow) which are embedded in electron-translucent material. (a, x36 000; b, x99 000)
Fig. 2. Acid phosphatase activity in collagen-containing vacuoles (arrows) of a fibroblast of the periodontal ligament of the mouse molar (see Beertsen et al., 1978). (x8000)
232
EVERTS et al. diographically and found that labelling was restricted to vacuoles containing collagen precursor molecules. Intracellular cross-banded collagen fibrils were unlabelled. Based on this evidence it was concluded that cross-banded collagen fibrils enclosed in lysosomal vacuoles of fibroblasts represent collagen that has been phagocytosed from the extracellular space.
Different connective tissues contain different levels of internalized collagen
Fig. 3. Collagen-containing vacuoles in a periosteal fibroblast. Note the presence of broad cross-banded collagen fibrils (arrows) which are formed by aggregation of single fibrils, and compare these with the relatively thin single fibrils (arrowheads) in the extracellular space (E). (x39 000)
The phenomenon that recently synthesized collagen is immediately broken down seems to occur only when the protein is not properly formed (Berg et al., 1980, 1983). Thus, it would seem plausible that the more collagen is synthesized (e.g. in tissues with a high turnover) the higher will be the chance of abnormal molecules being formed, and the higher the incidence of intracellular breakdown of newly synthesized collagen. However, it is questionable whether the cross-banded collagen fibrils found intralysosomally do represent this pathway. First, procollagen molecules are secreted into the extracellular space after which the formation of cross-banded fibrils occurs following cleavage of the terminal N- and C-propeptides of the procollagen molecules (reviewed by Kielty et al., 1993a). Second, by using explants of soft connective tissue it was demonstrated that blocking the synthesis of collagen had no effect on the appearance of intracellular cross-banded collagen fibrils in the lysosomal apparatus of the fibroblasts (Everts et al., 1985b). Subsequently it was shown that colchicine, a compound which strongly interferes with secretion of newly synthesized collagen precursors, had no influence on the amount of intracellular cross-banded collagen fibrils either in vivo (Beertsen et al., 1984) or in vitro (Everts & Beertsen, 1987). Third, incubations carried out with the phagocytosisblocking agent cytochalasin B resulted in an almost complete absence of fibrils in the vacuolar apparatus (Everts et al., 1985b, 1989). Finally, Marchi and Leblond (1983) studied the fate of collagen precursors autora-
Intracellular collagen is found in all soft connective tissues. However, considerable differences exist between various tissues (Svoboda et al., 1981). To date the highest amount of intracellular collagen is found in those tissues which are characterized by a rapid collagen turnover, such as the periodontal ligament and gingiva (Beertsen & Everts, 1977; Frank et al., 1977; Beertsen et al., 1978; Svoboda & Deporter, 1981; Svoboda et al., 1981; Deporter et al., 1984; Hirashita et al., 1985). Lower levels are found in tissues with a slower turnover such as skin (Svoboda et al., 1981).. Comparisons between various tissues indicate not only that there are tissue-related differences in the amount of intracellular collagen, but also that remarkable differences appear to exist within the same tissue. An example is provided by the periodontal ligament of the continuously erupting mouse incisor. This tissue forms the connection between the root and the inner wall of the socket in the alveolar process, and provides anchorage for the tooth. During eruption, the ligament that attaches to the tooth moves along with the incisor in the direction of the oral cavity (at a rate of approximately 1 mm per week), whereas the part anchored in the alveolar bone remains stationary. Between these two compartments of the ligament a zone of shear exists (Beertsen et al., 1974, Beertsen, 1975). A seven-fold higher amount of intracellular collagen is found in this zone of shear (the mid-region of the ligament) (Fig. 4; Beertsen & Everts, 1977). Also, in the lamina propria of the gingiva, differences exist in levels of intracellular collagen among various sites. The tissue underneath the outer oral epithelium contains far less phagocytosed collagen than that in cli~se proximity to the tooth (Svoboda & Deporter, 1981). Another example of site-specific differences is found in cultured periostea. Periosteal fibroblasts of long bones contain 5-fold less intracellular collagen fibrils than those presen t in the periosteum of calvariae (Everts et al., 1991). Since, in this in vitro study, the tissue samples were obtained from the same animals and cultured under the same conditions, the data indicate tissuespecific differences in phagocytic behaviour of fibroblasts. Isolated cells from different origins also appear to express differences in this respect (McCulloch & Knowles, 1993).
Phagocytosis and intracellular digestion of collagen
233
Collagen phagocytosis
in P D L
% collagen-containing vacuoles 0,8
0,6
0,4
0,2
i1-1
o1-1
il-2
ol-2
TRP
ARP
Fig. 4. The volume density of collagen-containing vacuoles in the periodontal ligament (PDL) of the mouse incisor. The values represent the mean % +_SD. TRP: tooth-related part of the ligament; ARP: alveolar bone related part of the ligament (o1: outer layer of the TRP, adjacent to the ARP; il: inner layer, adjacent to the tooth). (Data derived from Beertsen & Everts, 1977).
Fibroclasts Several authors have mentioned the presence of connective tissue cells which are characterized by the presence of very large numbers of collagen-containing vacuoles (Fig. 5; Garant, 1976; Beertsen et al., 1978; Bauer et al., 1979; Schellens et al., 1982; Fell et al., 1986; Ljuhin et al., 1992). Some of these cells are found specifically in close vicinity to bone-lining osteoclasts (Garant, 1976; Beertsen et al., 1978; Rifkin & Heijl, 1979), and are considered to play a role in the removal of collagen freed from the bone by the osteoclasts, or in the digestion of Sharpey's fibres loosened during bone resorption (Heersche, 1978). These cells differ from 'normal' fibroblasts in that: (i) the number of ingested collagen fibrils is much higher, (ii) the vacuoles which enclose the fibrils are almost exclusively of the translucent type, and (iii) the majority of the vacuoles contain only one or two fibrils (Fig. 5). Given the high number of fibrils taken up by these cells, some authors called them fibroclasts (Garant, 1976; Beertsen et al., 1978; Bauer et al., 1979). Fibroclast-like cells have been found in a variety of locations, e.g. in rodent teeth just below the apical termination of the junctional epithelium (Fig. 6) where the tooth-side of the ligament is detached from the tooth and completely broken down (Schellens et al., 1979, 1982), in the uterus during involution (Inouye et al., 1983), in the tail fin of metamorphosing tadpoles (Usuku & Gross, 1965; Takahama et al., 1992), after
topical application of lipopolysaccharide to the gingival epithelium of rat molars (Ljuhin et al., 1992) and after treatment of minced synovial tissue with NaF or dibutyryl-cAMP (Fell et al., 1986). The frequency of occurrence of fibroclasts has not yet been established. However, in cultured periosteal tissue explants (Everts et al., 1985b, 1989; Everts & Beertsen, 1987), it appears that this cell type can be found rather easily, making up approximately 5-10% of all fibroblasts. Whether the fibroclast constitutes a different subset of fibroblasts or represents just another stage in the life-cycle of a 'normal' fibroblast remains to be established.
Enzymes involved in the phagocytosis of collagen DIGESTIONPRIORTO UPTAKE Matrix metalloproteinases From the frequent presence of fibrillar fragments of limited length in the lysosomal apparatus of connective tissue cells, it is generally concluded that prior to internalization, partial digestion of the fibril a n d / o r its associated molecules occurs (Etherington, 1977; Reynolds, 1986). The events occurring at this stage are only partly understood. Several authors have suggested that collagenase plays a role in such an initial attack (e.g. Harris & Cartwright, 1977). However, so far no direct evidence
234
EVERTS et al.
Fig. 5. Fibroclasts in periosteum of rabbit calvaria. Note the large number of collagen fibrils (arrowheads) enclosed in electron-translucent vacuoles. (x20 000) has appeared in the literature supporting the view that the enzyme is indeed involved. In contrast, some findings shed doubt on the role of collagenase in this process. By using an in vitro model the effect of two compounds known to interfere with the activity of matrix metalloproteinases was investigated on the uptake of collagen fibrils by fibroblasts (Everts et al., 1989). The compounds used were an antibody against collagenase, which strongly inhibited the activity of the enzyme (Hembry et al., 1986), and Tissue Inhibitor of Metalloproteinases I (TIMP-1). Although it was shown that the antibody could penetrate the tissue, in none of the experiments was there an effect on the phagocytosis of collagen fibrils (Everts et al., 1989). A lack of effect was also noted with TIMP-1, a compound with high affinity for collagenase and having the ability to penetrate tissue (Mignatti et al., 1986). Not only exogenous TIMP added to the explants, but also endogenous inhibitor synthesized by the tissue itself (Everts et al., 1993) failed to have an effect on collagen phagocytosis (Everts et al., 1989, 1991). Taken together, these data strongly suggest that collagenase does not play a crucial role in the process leading to uptake and subsequent digestion of collagen fibrils. Consistent with these data is the observation that the uptake of collagen-coated beads by cells is not affected after pretreatment of the beads with collagenase (Knowles et al., 1991).
Further support for the view that collagenase is not a key enzyme in the process of collagen phagocytosis comes from studies in which the level of endogenous collagenase was modulated by cytokines and growth factors. Addition of interleukin lcz to periosteal explants, alone or in combination with epidermal growth factor, resulted in a 10-100 fold increase of the release of procollagenase (Van der Zee et al., 1993, 1994), both in the conditioned medium and in the tissue itself (Van der Zee et al., 1994). Transforming growth factor [3, on the other hand, decreased release. Assessment of the amount of phagocytosed collagen under these conditions revealed that interleukin lcz combined with epidermal growth factor had an inhibiting effect on phagocytosis whereas this parameter was increased by transforming growth factor [3. Thus, an inverse relationship exists between the level of procollagenase and the amount of internalized collagen (Table 2; Van der Zee et al., 1995). Further support for the importance of the intracellular route was the positive correlation between phagocytosed collagen and the actual amount of collagen broken down as assessed by hydroxyproline release (Van der Zee et al., 1995). Although these data support the view that collagenase is not of crucial importance in the digestion of phagocytosed collagen fibrils, it certainly.does not exclude a possible involvemer~t of other members of the
Phagocytosis and intracellular digestion of collagen
235 Table I
Phagocytosis Collagenase Hydroxyproline Vvcf/fib mUm1-1 ~gm1-1
Fig. 6. Engulfment of collagen fibrils (arrowheads) protruding from acellular cementum (C) covering the incisor of a mouse. The fibroblast dislodges collagen fibrils from the tooth in the gingival area of the erupting incisor. In this area the part of the ligament which moves with the erupting tooth in the direction of the oral cavity (Beertsen et al., 1974) is completely broken down just apical of the termination of the junctional epithelium (Schellens et al., 1982). (x76 000) matrix metalloproteinase family. In fact, recent studies performed in our group have d e m o n s t r a t e d positively the involvement of this class of enzymes. By using a low molecular weight inhibitor that selectively blocks the activity of the matrix metalloproteinases (Delaiss6 et al., 1985), the .uptake and subsequent intracellular digestion of collagen fibrils ceased in periosteal tissue explants (Creemers et al., 1994). C o m p a r a b l e effects (Creemers et al., 1994) were f o u n d b y using selective inhibitors for gelatinases (MMP-2 and MMP-9) and stromelysin (Hill et al., 1994a). As gelatinase A (MMP-2) is found in almost all tissues, and this e n z y m e appears to have the capacity to digest partially native type I collagen (Tournier et al., 1994; Aimes & Quigley, 1995), we suggest tentatively that m e m b r a n e - b o u n d gelatinase A is involved in extracellular digestion of collagen fibrils prior to phagocytosis. If gelatinase A does indeed participate in this, the question arises w h y TIMP-1 had no effect on phagocytosis (see above). One explanation could be that the inhibitor is not v e r y effective at inhibiting gelatinase A ( H o w a r d et al., 1991). An alternative explanation is that the site of activity of the e n z y m e - at the plasma m e m b r a n e - is not freely accessible to the inhibitor.
Experiment A Control PDGF TGF-~ IL-1R IL-lc~+PDGF IL-I~+TGF-~
4.5_+1.8 4.4_+1.3 7.4_+1.2 1.1_+0.9 1.2_+1.0 3.7_+3.0
16_+2 18_+2 14_+3 92_+31 119_+49 49_+24
ND ND ND ND ND . ND
Experiment B Control EGF IL-lc~ EGF+IL-lc~
8.5_+3.5 7.9_+4.6 3.3_+3.4 1.8_+1.5
10_+6 12_+7 61_+35 186_+40
3.3_+0.9 2.9+1.1 1.4_+0.4 0.7_+0.2
Periosteal explants were cultured for 72 h in the absence or presence of platelet-derived growth factor (PDGF, 10 ng ml-1), transforming growth factor ~ (TGF-~, 10 ng ml-1), interleukin 1~ (IL-lc~, 10 ng m1-1) and/or epidermal growth factor (EGF, 10 ng ml-1; see Van der Zee et al., 1993, 1994, 1995). The amount of phagocytosed collagen was assessed morphometrically and is expressed as the volume density of collagen fibrils per fibroblast (Vvcd). The level of collagenase (latent and active) was determined by using radiolabelled fibrillar collagen as substrate (see Everts et al., 1991). The amount of hydroxyproline released in the medium during culturing was established according to the method described by Van der Zee et al. (1995). All values represent mean + SDof at least 6 cultured explants. ND: not determined. Statistical analysis revealed the following significant results: (a) Transforming growth factor ~ enhanced phagocytosis but decreased the release of (pro)collagenase, (b) Interleukin la inhibited phagocytosis but increased the level of (pro)collagenase, (c) the level of (pro)collagenase was negatively correlated with both the amount of phagocytosed collagen and the amount of released hydroxyproline. (Data derived from Van der Zee et al., 1995.) Some indirect s u p p o r t for the i n v o l v e m e n t of gelatinase A comes from studies in which the effects of concanavalin A were investigated. This lectin strongly p r o m o t e s synthesis and secretion of gelatinase A b y fibroblasts (Overall & Sodek, 1990; Overall et al., 1991) and also significantly enhances phagocytosis of collagen fibrils or collagen-coated beads (Everts & Beertsen, 1992a; Knowles et al., 1991, respectively).
Cysteine proteinases Another class of e n z y m e s that have the capacity to digest fibrillar collagen is the cysteine proteinases, a m o n g which are cathepsins B, L, and N (Burleigh et al., 1974; Etherington, 1976; Maciewicz & Etherington, 1988). Interference with the activity of these e n z y m e s b y using a variety of selective inhibitors (E-64 and Z-PheAlaCH2F) which inhibit cysteine proteinases in the extracellular e n v i r o n m e n t as well as intracellularly after
236 uptake by the cell, did not seem to affect uptake of collagen (Everts et al., 1985b; Van Noorden & Everts, 1991), thus suggesting that internalization of collagen fibrils does not require an extracellular activity of cysteine proteinases. Serine and aspartic proteinases Far less is known about the possible contribution of the other two classes of proteolytic enzymes: the serine and aspartic proteinases. Using inhibitors of these proteolytic enzymes, no effects on the uptake of fibrillar collagen have been noted so far (Everts & Beertsen, 1988).
DIGESTIONFOLLOWINGUPTAKE The digestion of fibrillar collagen, once it has been taken up by the fibroblast and brought into contact with the lysosomal apparatus, depends on the activity of cysteine proteinases (Everts & Beertsen, 1988, 1992b). This conclusion is based on the finding that inhibitors of cysteine proteinases (see the section on Cysteine Proteinases) have dramatic effects on the presence of intracellular fibrillar collagen; an increase of up to 100-fold in intracellular collagen is seen within a culture period of 48 h (Fig. 7; Everts et al., 1985b, 1989; Everts & Beertsen, 1987; Van Noorden & Everts, 1991). Similar effects were noted (L. Creemers, K. Hoeben, W. Beertsen & V. Everts, unpublished observations) using inhibitors (Ep453, CA074Me) that block the activity only intracellularly (Buttle et al., 1992a,b; Hill et al., 1994b). The accumulation of intracellular collagen was associated with a decrease of collagen breakdown products in the conditioned medium (Creemers et al., 1994). Although
EVERTS et al. these observations indicate participation of cysteine proteinases in the lysosomal digestion of the collagen fibrils taken up by the cells, it is not entirely clear whether the enzymes are primarily responsible for the digestion of the collagen fibril itself, or for the degradation of the (non)-collagenous proteins associated with it (see below). The nature of the cysteine proteinase(s) involved has not been elucidated completely yet. Several members of this proteinase family, including cathepsins B, L and N, have the capacity to degrade collagen, the most active one being cathepsin L. By studying the effect of a selective inhibitor of cathepsin B, Van Noorden and Everts (1991) were able to show blockage of collagen digestion comparable to that seen with a general inhibitor, thus indicating that at least cathepsin B is involved.
Role of (non)-collagenous proteins surrounding fibrillar collagen Fibrillar collagen in the extracellular space is surrounded by, or even embedded in, a meshwork of (non)-collagenous proteins, among which are proteoglycans, glycoproteins, and collagen types V and VI. During uptake some of these components remain associated with the fibril and bridge the space between the collagen and the membrane lining the phagosome (Fig. 8; Melcher &Chan, 1981; Everts & Beertsen, 1992b; Dingemans & Teeling, 1994). One might assume that they play a role in the chain of events leading to recognition, which appears to be integrin-mediated (McKeown et al., 1990), and to subsequent uptake of fibrillar
Fig. 7. Numerous collagen-containing vacuoles (arrows) in a fibroblast of periosteal tissue (obtained from rabbit calvaria) cultured for 48 h in the presence of the cysteine proteinase inhibitor E-64 (Everts et al., 1985). (x25 000)
Phagocytosis and intracellular digestion of collagen
237
Fig. 8. Collagen-containing vacuole of the electron-translucent type stained with polyethyleneimine for the presence of proteoglycans. Note the electron-dense precipitates (arrowheads) surrounding the enclosed fibril. (Micrograph kindly donated by K. Dingemans, see Dingemans & Teeling, 1994). (x49 000) collagen. Numerous extracellular matrix proteins contain integrin-recognizable RGD-sequences including several collagens (e.g. types I, III, V and VI) and glycoproteins such as fibronectin (see Edelman & Crossin, 1991; Bosman, 1993). Under normal conditions these fibril-associated components are likely to create a three-dimensional meshwork in which fibrils are embedded and bundled. As in almost all instances phagocytosis involves uptake of only one or at most a few fibrils, loosening of the fibrils from a bundle is likely to occur, although it has never been observed as such. H o w the cell accomplishes this is unknown, but perhaps the work of Chen and coworkers (Chen et al., 1984; Mueller & Chen, 1991) might give some clues. In their studies it was shown that, on the membrane of transformed cells, relatively high levels of a matrix-degrading enzyme were present at focal sites (Kelly et al., 1994). These enzyme-rich patches were frequently found on cellular extensions, the so-called invadopodia, which were shown to induce breakdown of extraceltular matrix (Kelly et al., 1994). Since their studies indicate that the membrane-bound enzyme is a 72 kDa metalloproteinase (Monsky et al., 1993), probably gelatinase A (Ward et aI., 1991, 1994; Murphy et aL, 1992), it is tempting to suggest that it is indeed this enzyme that attacks the fibril initially prior to internalization (see also the section on Matrix metalloproteinases). At this stage not all fibril-associated components are lost. Some of them are broken down after the uptake has occurred (Dingemans & Teeling, 1994), probably under the influence of cysteine proteinases (Nguyen et al., 1990; Buttle et al., 1991). Summarizing, we propose the following sequence: fibroblast extensions attach to extracellular matrix ligands through their integrin receptors (McKeown et al., 1990). This binding initiates a cascade of events, including a rearrangement and reorganization of elements of the cytoskeleton, in particular the microfilament system (Isberg & Tran Van Nhieu, 1995) as well as an increased
expression and release of gelatinase A (Larjava et al., 1993). Cytoplasmic extensions surround the fibril and a microenvironment is created between the engulfing membrane and the enclosed fibril. In this milieu membrane-bound enzyme (presumably gelatinase A) partially digests the collagen fibril a n d / o r its associated components. This action results in fragmentation of the fibril and its uptake into a phagosome. Lysosomes then fuse with the phagosome, and further breakdown is accomplished in an acidified environment under the influence of cysteine proteinases. Given the ubiquitous occurrence of collagen phagocytosis by fibroblasts, these cells may be considered 'professional phagocytes' (Rabinovitch, 1995).
Intracellular degradation of different types of collagen Besides fibrillar (type I and III) collagen, type VI collagen is moderately abundant in virtually all connective tissues (Hessle & Engvall, 1984; Engvall et al., 1986; Trueb et al., 1987). It surrounds fibrillar collagen and may be involved in the attachment of cells to the extracellular matrix (Keene et aI., 1988; Aumailley et al., 1989). In spite of its ubiquitous occurrence, surprisingly little is known about the w a y in which type VI collagen is degraded (see Murphy & Reynolds, 1993). It is one of the few known collagen types that is not digestible by collagenase. Recently, however, Kielty and co-workers (1993b) showed that partial degradation occurs by the action of serine proteinases. During our in vivo and in vitro studies on the digestion of fibrillar collagen we observed the presence of filamentous material in the lysosomes of fibroblasts characterized by a crossbanding with a periodicity of about 100 nm (Fig. 9). In accordance with data presented by Bruns et al. (1986), these cross-banded structures were shown to consist of type VI collagen. Interference with collagen synthesis did not affect the amount of intracellular type VI colla-
238
EVERTS et al.
Fig. 9. Collagen-containing vacuoles in a fibroblast of calvarial periosteum cultured for 24 h in the presence of the cysteine proteinase inhibitor E-64 in combination with the acidotropic agent NH4C1 (Everts & Beertsen, 1992; Everts et al., 1994b). Arrow indicates broad-banded type VI collagen in a lysosomal vacuole which also contains fibrillar collagen (arrowhead). (x48 000)
gen, whereas inhibition of phagocytosis by cytochalasin B completely prevented its appearance, thus indicating that fibroblasts can internalize this type of collagen. It was shown subsequently that digestion of this collagen depends on the participation of several enzymes: initially serine proteinases, probably in combination with collagenase, and at a later stage pH-sensitive lysosomal enzymes (Everts & Beertsen, 1992b; Everts et al., 1994b). In addition to type I, III and VI collagen, Knowles et al. (1991) have demonstrated that fibroblasts can recognize and internalize latex beads coated with type V collagen. Thus the data presented so far in the literature suggest that numerous collagen types are susceptible to phagocytosis and subsequent digestion by connective tissue cells.
hance phagocytosis. In combination, both cytokines were antagonistic. Notably, the combined effect of interleukin lc~ and transforming growth factor [3was only found at later time intervals (after 72 h) (Van der Zee et al., 1993, 1994, 1995), suggesting the existence of an indirect effect, not yet understood. In addition to cytokines, certain hormones may modulate the intracellular pathway of collagen digestion; for example, phagocytosis of collagen in cultured pig synovial tissue decreases under the influence of cortisol (Fell et al., 1986), and during post-partum involution of the uterus high levels of phagocytosed collagen are found (Schwarz & G61dner, 1967; Brandes & Anton, 1969; Okamura et al., 1976; Dyer & Peppler, 1977; Inouye et al., 1983). Certain nutrients (Svoboda et al., 1979b) and calcium (McCulloch & Knowles, 1993) also appear to influence the phagocytic pathway.
Modulation of collagen phagocytosis Given the remarkable heterogeneity among and within tissues with respect to collagen phagocytosis (see the section: Different connective tissues contain different levels of internalized collagen), one is inclined to accept that this activity is modulated somehow, presumably by growth factors and cytokines, compounds known for their pronounced influence on numerous cellular activities (reviewed by Dueul, 1987; Sporn & Roberts, 1988; Nathan & Sporn, 1991; Birkedal-Hansen, 1993). This assumption was investigated in an in vitro model system consisting of periosteal tissue explants, using a variety of growth factors (fibroblast growth factor, epidermal growth factor, platelet-derived growth factor, and transforming growth factor [3) and the cytokine interleukin 10~ (Van der Zee et al., 1995). Of these compounds, only transforming growth factor [3 and interleukin 10caffected phagocytosis. As already mentioned, interleukin lcz decreased uptake of fibrillar collagen, whereas transforming growth factor 13appeared to en-
Collagen phagocytosis and disease Phagocytosed collagen is not only found in healthy tissues, but also under several pathologic conditions such as in certain tumours, Hurler syndrome, epidermolysis bullosa, and arthritis (reviewed by Renteria & Ferrans, 1976). However, "as in none of the studies have quantitative data been provided, and no information was available on the turnover and remodelling in the tissues studied, it remains to be seen whether these observations hold clues to the importance of the intracellular collagen breakdown in pathologically altered tissues. Fibrosis has been evaluated in somewhat more detail. Hall and Squier (1982) as well as McGaw and Porter (1988) found that fibrosis-inducing agents (phenytoin and cyclosporin) were associated with a decreased amount of phagocytosed collagen. In line with these in vivo data are in vitro observations (McCulloch &
Phagocytosis and intracellular digestion of collagen Knowles, 1993) showing that two fibrosis-inducing drugs (dilantin and nifedipine) inhibit phagocytosis of collagen-coated beads by fibroblasts.
Role of collagen phagocytosis in collagen turnover The finding, that in all connective tissues studied so far fibroblasts are found containing cross-banded collagen fibrils in their lysosomal apparatus, prompted several authors to suggest that during normal turnover of collagen the intracellular pathway is an important route (Ten Cate, 1972; Beertsen et al., 1974; Ten Cate et al., 1976; Beertsen & Everts, 1977; Svoboda et al., 1981). Although direct proof for this assumption is still-lacking, there is considerable indirect evidence in support of this view. First, by comparing various soft connective tissues, a positive correlation is found between the amount of intracellular collagen and the rate of collagen turnover as determined biochemically (Svoboda et al., 1981). Second, calculation of the digestion time of internalized collagen (approximately 30 min; Everts et al., 1989) suggests a half-life of collagen which corresponds with the turnover time as assessed biochemically (Everts & Beertsen, 1988; Everts et al., 1989). In 1988 we and others (Everts & Beertsen, 1988; Sodek & Overall, 1988) proposed that two pathways of collagen digestion could be discerned: (i) a collagenaseindependent intracellular route, and (ii) a collagenasedependent extracellular pathway. The first route would be important during normal turnover, and the collagenase-mediated one when large amounts of collagen are broken down accompanied by a loss of tissue architecture (e.g. inflammation and involution of the uterus). Support for the view that, under physiological conditions, the collagenase-mediated route of collagen digestion is less important than formerly thought, is presented by recent studies in which collagen remodelling was assessed in mutant mice characterized by collagenase-resistant type I collagen (Liu et aI., 1995; Krane, 1995). Growth and development of these mice appeared to occur normally up to young adulthood, a time period in which an extensive turnover and remodelling of all connective tissues takes place. Disturbances in remodelling were only noted with increasing age. In the older animals fibrosis of the dermis, as well as an impaired postpartum involution of the uterus, was noted.
239 to be elucidated. As connective tissue turnover and remodelling is considered to depend on this intracellular route, future research, should include the modulation of this pathway by compounds such as growth factors and cytokines. In exploring this field the remarkable differences among various tissues with respect to the level of phagocytosis may be of help. Questions such as which factors initiate phagocytosis, and how a fibril is recognized by a fibroblast, should be addressed.
Conclusion In summary, we propose (Fig. 10) that under physiological conditions characterized by an equilibrium between synthesis and degradation, the intracellular pathway is the major route of collagen digestion (Everts & Beertsen, 1988; Sodek & Overall, 1988). The extracellular (collagenase-mediated) degradation occurs when, in a relatively short time interval, large amounts of collagen have to be broken d o w n (e.g. inflammation and involution of the uterus). Phagocytosis of collagen is considered to include the following steps: cytoplasmic extensions of the fibroblast probe their environment, surround and enclose part of a collagen fibril and segregate this from the extracellular environment. Proteolytic enzymes belonging to the family of matrix metalloproteinases (possibly gelatinase), which are attached to the membrane, are activated in the segregated area and exert their activity. After partial breakdown of the fibril, including some of its surrounding collagenous and non-collagenous proteins, the fragmented collagen fibril is taken up by the cell. This process is mediated by cytokines such as interleukin lc( and transforming growth factor [~. At the site of uptake microfilaments are present at high concentrations, thus providing the contractile force needed for segregation as well as internalization. The solitary fibril fragment is subsequently enclosed in a phagosome, after which fusion with lysosomal vacuoles takes place. Several phagolysosomes fuse and form large structures containing a number of fibrils. The first step in the subsequent digestion involves breakdown of the proteinaceous core surrounding the fibril, which then results in aggregation of the single fibrils into thicker fibrillar structures. Finally, these structures are degraded by enzymes such as cysteine proteinases, including cathepsins B and L.
New directions Since the first description of fibrillar collagen enclosed in lysosomal vacuoles by Luse and Hutton (1964), our knowledge of the role and mechanisms of this pathway of collagen digestion has increased considerably. However, many details of the processes involved still remain
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Fig. 10. Schematic presentation of the intracellular and extracellular pathways of collagen digestion. Under equilibrium conditions the intracellular pathway is considered to be the most important route of degradation. Cross-banded collagen fibrils with their associated (non-) collagenous proteins are engulfed by fibroblast extensions. Membrane-bound enzymes (probably gelatinase A) partially digest the fibril after which it is taken up by the fibroblast and further degraded in the lysosomal apparatus by cysteine proteinases (CPs). Digestion of relatively large amounts of collagen occurs primarily in the extracellular space under the influence of a variety of enzymes of which the matrix metalloproteinases are likely to be the most important ones. GLA: gelatinase A (MMP-2); GLB: gelatinase B ( MMP-g); CL; collagenase (MMP-1); SL: stromelysin (MMP-3). broblastic tumor: light and ultrastructural study of a case. Hum. PathoL 4, 419-29. AUMAILLEY, M., M A N N , K., VON DER MARK, H. & TIMPL, R. ( 1 9 8 9 ) Cell attachment properties of collagen type VI and Arg-GlyAsp dependent binding sites c~2(VI) and R3(VI) chains. Exp. Ceil Res. 181, 463-74. A Z N A V O O R I A N , S., MURPHY, A. N., STETLER-STEVENSON, W. G. &
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