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1. Systemic changes in inflammation - The acute phase response
2. Local changes in inflammation
3. Mediators of local changes-eicosanoids
4. Mediators of local changes - Cytokines
5. Interleukin-1 (IL-1)
6. Tumour necrosis factor (TNF)
7. Interferon gamma (IFNg)
8. Interactions of osteotropic influences
References
Discussion
T.M. Skerry
Department of Anatomy, School of Veterinary Science, University of Bristol, Southwell Street, Bristol BS2 8EJ, UK
The inflammatory response is a process which forms a defence against the effects of trauma or invasion by foreign organisms or substances. Inflammation is mediated by local and systemic changes in expression of its activators and inhibitors. Many of these regulators of inflammation are also the controllers of normal cellular activity, so the effects of inflammation are due to changes in the amounts, ratios and timings of their expression, rather than de novo expression of specific mediators of inflammation.
The effects of inflammation on bone growth are two-fold. Firstly, systemic inflammatory effects have consequences on hormone, mineral and nutrient metabolism which affect bone growth. Secondly, cytokine mediators of inflammation cause local changes in cell regulation to influence both endochondral processes in the growth plate, and modelling and remodelling activity associated with appositional growth.
The complexity of the osteotropic influences responsible for maintaining normal bone development mean that inflammatory process will have different and unpredictable effects on linear growth at different times, and under different nutritional, biochemical, physical and psychological circumstances. This review will examine the effects of systemic and local changes in inflammation which have effects on bone growth and remodelling, focusing on systemic effects of the acute phase response, and the local actions of the eicosanoids and three cytokines with particularly potent actions on the metabolism of cells in bone and cartilage.
The major systemic effect of inflammation which has repercussions on the growth of bone is the acute phase response (APR). This process is one which appears to have a protective function for the organism (McGlave, 1990). Varying degrees of local inflammation, whether caused by trauma, infection or other stimuli give rise to a series of changes in the circulating concentrations of the so-called acute phase proteins. These include proteins with coagulation and complement system functions, their inhibitors, transport proteins and a miscellaneous group including C-reactive protein (CRP). In addition, the APR is associated with changes in some hormones (insulin, glucocorticoids, and catecholamines) (Adam) et al., 1987), vitamins (Louw et al., 1992), and minerals-primarily iron and zinc (Boosalis et al., 1992), although the latter may result from the changes in transport protein levels. There is also activation of proteolytic enzyme cascades connected with clotting, complement, kinin and fibrinolytic pathways, and a change in amino acid metabolism, with catabolism of muscle protein, and transport to the liver.
The APR appears to be linked to elevated levels of circulating interleukin-1 (IL-1), which influences hepatocyte metabolism to elevate levels of the acute phase proteins. However, infusions of IL-1 are less effective in stimulating the acute phase response than that caused by an acute inflammatory stimulus, so it is clear that this cytokine is not the sole mediator of the response (Lewis, 1986). There is evidence that other cytokines may be elevated in the acute phase response (Wegenka et al., 1993; Grunfeld & Feingold, 1992; Mazlam & Hodgson, 1992), but it is not yet clear whether this is a cause of the response or a part of it. In graft versus host disease (GVHD), the systemic inflammatory response to challenges such as those seen in bone marrow transplantation, circulating levels of the cytokine tumour necrosis factor alpha (TNFa) are increased (Hirokawa et al., 1989). Like IL-1, this cytokine has potent but complex effects on bone growth and remodelling which are discussed later. In addition, interferon-gamma (IFNg), also discussed later, has effects on the acute phase response, as it regulates the complement proteins C2 and C4 (Strunk et al., 1986).
The effect of the APR on linear bone growth is to reduce growth velocity and ultimate bone length. In GVHD following bone marrow transplantation for leukaemia, both boys and girls were found to have significantly reduced growth velocity, when compared with matched groups who failed to develop the inflammatory response (Shinohara et al., 1991) (Fig. 1). This indicates that the net effect of a systemic inflammatory response is to slow bone growth. Clearly reductions in circulating insulin and zinc would be expected to have that effect, but it is at odds with reduced levels of glucocorticoids. Since the magnitude of the acute phase response is itself attenuated by plasma protein deficiency (Jennings, Bourgeois & Elia, 1992), it is clear that nutritional status can influence the effect of concurrent systemic inflammation on growth. The net effect of systemic inflammatory responses on bone growth is therefore a balance between opposing influences on growth, which results in a retarding effect on the endochondral process.
Locally, the mechanisms underlying the effects of inflammation on bone are more complex. In specific examples, growth may be either retarded or enhanced by such stimuli. For example, inflammation caused by minor trauma to the distal ulnar growth plate of growing dogs and horses causes either retardation or cessation of growth. (This growth plate is susceptible to those injuries because of its superficial nature and conical conformation, both of which predispose it to shearing injuries with minor force.) However, among treatments for the shortening, valgus and varus deformities which result, is surgical incision of the periosteum overlying the region with delayed growth (Adams, 1987). Following this procedure, the growth rate increases, and leg lengthening resumes with correction of the deformity. The mechanism by which the inflammation associated with surgically induced trauma not only restores normal growth plate function but also corrects deformity is unknown.
To understand the possible
mechanisms of local control of bone growth in response to inflammation, it is necessary to
consider the regulation of normal bone growth and remodelling by interactions between
hormones, cytokines and mechanical forces. Much of that information is covered elsewhere
in this book, so attention will be focused only on those processes where they link with
changes specifically associated with inflammation.
Among the many mediators of the inflammatory response are the eicosanoids - the arachidonic acid derivatives which include prostaglandins, and the 'inflammatory' cytokines with local actions on chondrocytes and bone cells.
The eicosanoids are derived from arachidonic acid, which in turn is manufactured from membrane phospholipids, and can therefore be made in a wide range of cells (Fig. 2). Arachidonic acid is converted into the prostaglandins and thromboxanes by the action of cyclo-oxygenase, and into leukotrienes by lipoxygenase. Many studies have shown roles for the prostaglandins and their derivatives in the control of bone remodelling (Saito et al., 1990; Das, 1991). However, the process of expression of the eicosanoids is subject to a complex series of regulatory steps, which are altered in response to inflammation (Hopkins, 1990).
The conversion of the phospholipids to arachidonic acid involves phospholipase A2, which is in turn affected by other factors. This action can be regulated in both directions, positively by mechanical loading (Binderman et al., 1988) and negatively by lipocortins. The lipocortins are a family of glucocorticoid-induced proteins, with a range of sizes from 15,000 to 200,000 Daltons. However, despite this wide variation in size, they exhibit remarkable cross reactivity with monoclonal antibodies, and a strong functional identity (Bowen & Fauci, 1993). Since glucocorticoid metabolism is altered in the acute phase response, it is clear that regulation of eicosanoid synthesis can play an important role in bone lengthening.
The actions of eicosanoids on bone growth and remodelling are to affect both chondrocytes in the germinative layer of the epiphyseal plate, and to modulate bone remodelling and the responses of bones to other stimuli.
Growth plate chondrocytes are not only responsive to prostaglandins, but they exhibit different responses to different members of the eicosanoid family (O'Keefe et al., 1992). It can be seen (Fig. 3) that prostaglandins E1 and E2 are potent stimulators of both chondrocyte thymidine incorporation and cyclic AMP production, which is less affected by prostaglandins A1 and F2a
Although those experiments showed effects of prostaglandins on markers of replication and second messenger response in cultured cells, it appears that the mechanisms involved share common features with the effect of eicosanoids on bone remodelling. Mechanical loading in vivo has been shown to affect growth plate thickness and ultimate bone length, and in a similar model of endochondral ossification, differential expression of prostaglandins. Good-ship & Oryan (1993) showed that in sheep, mechanical loading affected the proximal tibial growth plate, increasing bone length, and mineralization rate. Similar mechanical regimes regulated expression of prostaglandins E2 and F2a differentially in the zone of endochondral ossification of a healing osteotomy in the tibiae of sheep. Mechanical loading regimes which resulted in improved rate of healing were associated with greater increases in prostaglandin E2 than prostaglandin F2a, while regimes which resulted in hypertrophic nonunion showed the reverse. The mechanisms of action of such effects is far from clear, but the increasing body of information on regulation of cellular activity by extracellular matrix components (Streuli et al., 1993), which are in turn affected by mechanical factors (Skerry et al., 1989; Skerry et al., 1990), represents one possible transduction system.
Although both chondrocyte replication and bone remodelling have been shown in vitro to be profoundly influenced by the actions of many cytokines (Goldring & Goldring, 1990), including those specifically affected by inflammatory responses, it is difficult to specify the relevance of such findings in vivo. This is because although the individual actions of osteotropic cytokines can be studied in vitro, in highly specified and characterised culture systems, such experiments lead to numerous different and often contradictory results. This apparent paradox has been suggested to be due to misconceptions in the consideration of targets for cytokine action (Nathan & Sporn, 1991). Instead of thinking of cell types as cytokine targets, it may be more appropriate to see tissues as targets for their actions. This would allow the action of a given cytokine to be seen as the sum of its effects as a soluble, membrane or matrix bound form, with effects on a total process within the tissue rather than only one aspect of a cell's metabolism.
With those reservations stated, it is clear that inflammatory processes influence both bone remodelling and linear growth (Shinohara et al., 1991). Many of the normal regulatory processes in endochondral bone formation and remodelling are associated with local expression of cytokines which are elevated by inflammatory processes (Fujita et al., 1990). In the following sections, three cytokines will be discussed in detail, to illustrate the way in which their part in the inflammatory process could influence bone growth and remodelling. The major effects of those cytokines on bone growth and remodelling are summarised in Fig. 4.
Fig. 4. Major cytokine
effects on bone growth and remodelling.
One of the cytokines first shown to have a positive action in stimulating bone resorption was IL-1. IL-1 exists in two forms (a and b), which share only limited sequence homology, but bind to the same membrane receptor (Dower et al., 1985). The mechanism of signal transduction following binding is not known, but follows internalisation of the ligand-receptor complex (Shen et al., 1990), and is associated with a rapid (30 second) rise in intracellular calcium (Catherwood, Onishi & Deftos, 1983).
The effects of IL-1 on bone growth are due to individual effects on chondrocytes and osteoblasts, while effects on remodelling will have the potential to influence the shape of the developing bones. Since chondrocyte prostaglandin E2 expression is reduced by IL-1 (Evequoz, Trechsel & Fleisch, 1985), and since prostaglandin E2 regulates chondrocyte cAMP, and cell proliferation (O'Keefe et al., 1992), it is clear that the cytokine has the ability to affect bone growth. However, since chondrocytes normally express IL-1 (Rash, Oronsky & Kerwar, 1988), it is clear that this effect is a result of overexpression, not the presence of a pathological mediator. Remodelling activity is similarly affected. Osteoblast proliferation is markedly down-regulated by IL-1 (Ohmori et al., 1988; Hanazawa et al., 1986), which has been shown to have potent effects in stimulating resorption in a number of systems.
IL-1 has powerful effects on cell cultures, stimulating synthesis of DNA and prostaglandin E2 and inhibiting alkaline phosphatase activity and collagen synthesis (Ikeda et al., 1988; Hurley et al., 1989). In cultured bone explants, IL-1 stimulates calcium release (Cochran & Abernathy, 1988), and increases in osteoclast number (Garrett & Mundy, 1989).
IL-1 enhances bone resorption in
vivo, in both man and animals (Konig, Muhlbauer & Fleisch, 1988; Ahn, Huang &
Abramson, 1990), and is currently thought to be the cause of most pathological bone
resorption in inflammatory diseases. In addition to enhancing osteoclast numbers, the
mechanism by which bone resorption is enhanced appears to be linked with the ability of
IL-1 to upregulate expression of integrin subunits. These molecules are involved in
osteoclastic attachment to bone matrix, and changes in their expression would therefore
influence the resorptive potential of those cells (Dedhar, 1989). It is interesting that
bones from different regions of the skeleton respond differently to the effects of IL-1
(Cochran & Abernathy, 1988), suggesting that there is location-dependent
susceptibility of skeletal cells to osteotropic influences, which could account for
regional differences in growth retardation and remodelling.
Although there is little structural similarity between the two cytokines, TNF shares many functions with IL-1. Like IL-1, there are two forms, TNF a and b, which share approximately 30% sequence homology. Two receptors exist for TNF, which bind both forms despite marked structural differences in the receptor proteins (Schall et al., 1990; Smith et al., 1990).
Like IL-1, TNFa is capable of influencing endochondral longitudinal bone growth. Differentiation of mesenchymal cells into chondrocytes is reversibly inhibited by TNFa (Yoshikawa et al., 1988; Hashimoto et al., 1989), and the cytokine decreases the synthesis of glycosaminoglycans by existing growth plate chondrocytes, and reduces their alkaline phosphatase activity (Enomoto et al., 1990; Centrella, McCarthy & Canalis, 1988). Administration of TNFa in vivo results in animals which are stunted (Yoshikawa et al., 1988).
Bone remodelling is also profoundly affected by TNF. As with IL-1, these effects have been shown in a number of model systems ranging from cell cultures through explants, and in vivo (Konig, Muhlbauer & Fleisch, 1988). However, the effects appear to be more variable than with IL-1, as different workers have found apparently contradictory results using similar model systems. Different groups have found no effect (Shapiro, Tatakis & Dziak, 1990; Nanes, McKoy & Marx, 1989), stimulation of proliferation (Gowen, MacDonald & Russell, 1988), or transient stimulation followed by inhibition (Canalis, 1987) or inhibition only (Yoshihara et al., 1990). These differences have been suggested to be due to 'postreceptor factors' (Weinberg & Larrick, 1987), which would appear to mean that other unknown influences modulate the responsiveness of cells to TNF. Such a mechanism appears to be a ubiquitous feature of most cytokines' actions.
TNF and IL-1 are stated to have
synergistic effects, in which suboptimal doses cause effects which are many times greater
than the sum of their individual activities (Stashenko et al., 1987). One possible
explanation for this is that TNF has been shown to induce IL-1 synthesis in endothelial
cells (Nawroth et al., 1986), which can then act on macrophages to induce further
TNF expression (Collart et al., 1986). While such amplification can have benefits
in the initial responses of the inflammatory system, with consequent benefits in dealing
with infections (Cross et al., 1989), it is clear that it also has the potential to
influence profoundly bone growth and remodelling.
Interferons were named because they were first discovered to interfere with viral replication. (Isaacs & Lindenmann, 1957). However, as with many cytokines, it has since become clear that the actions of interferon gamma (IFNg) are more diverse than the early discoveries suggested. Although IFNg is produced primarily by T cells, natural killer cells and macrophages have also been suggested as sources (Trinchieri et al., 1984).
In culture, IFNg is a potent inhibitor of bone resorption. In bone explants, it inhibits osteoclast differentiation (Vignery, Niven & Shepard, 1990), and reduces the activity of existing cells in a manner similar to calcitonin (Klaushofer et al., 1989). The actions of IFNg are highly specific. It has been shown to inhibit resorption stimulated by IL-1 and TNF. The actions on resorption stimulated by PTH or the active metabolite of vitamin D are more variable different groups finding no effect (Gowen, Nedwin & Mundy, 1986) or inhibition (Fuji) et al., 1990; Nanes et al., 1990). The mechanism of action of IFNg is not clear, as it has been shown that it is not associated with changes in receptors for IL-1 (Shen et al., 1990). It is likely that it involves the inhibition of synthesis and release of the metalloproteinases necessary for matrix degradation (Shen et al., 1988). In addition, IFNg inhibits DNA synthesis in cell cultures, an effect which is enhanced by co-incubation with TNF (Napes, McKoy & Marx, 1989).
The action of interferon in vivo is
in contrast with its clear ability to inhibit bone resorption in vitro. Although
there are limited numbers of experiments to support this, Epstein showed a lack of effect
of IFNg
on most markers of cyclosporin-induced bone loss in rats (Jacobs et al., 1992). In
addition, some parameters measured showed an increase in resorption. Vignery showed an
increase in numbers of osteoclasts in mice following IFNg treatment (Vignery, Niven &
Shepard, 1990), which is the opposite of the effect in vitro (Takahashi, Mundy
& Roodman, 1986).
The purpose of presenting the previous account of cytokine and eicosanoid actions is to illustrate the complexity of the control of bone and chondrocyte metabolism. It is unfortunate that experiments to determine the effects of cytokines produce such conflicting results in apparently similar but not identical conditions. However, this shows that the control of bone growth and remodelling is effected by a tightly regulated and highly specific sequence of ordered changes in expression of cytokines (Suva et al., 1993). This is not the sole manner of regulation of action of osteotropic agents. Modulation of receptors and inhibitors is a critical and important stage of regulation of action, which can completely reverse the effects of a given stimulus. The inhibitor for IL-1 is a normal circulating modulator of the actions of IL-1 (Seckinger et al., 1990), and its absence is capable of upregulating resorption or other effects of IL-1. Other inhibitors of cytokine actions include fragments of their receptors shed from cells, which bind the ligand without eliciting cellular responses.
Even after ligand receptor binding, there are many levels at which regulation of action is possible. Immediate intracellular consequences of binding such as tyrosine kinase activation, and proto-oncogene expression are both complex processes which involve many steps dependent on other processes within the cell. Even if all these actions occur, it is possible to express messenger RNA which will not be transcribed into protein, or protein which will either not be secreted, or which will be inhibited.
The effects of inflammation on bone growth are therefore hard to predict accurately, as the inflammatory process is one which involves changes in many agents with osteotropic actions. It is rare that any of these agents stimulate any long-term gain in bone length which is reflected in an increase in adult height, so the likely effect of inflammation is to reduce bone length, by a combination of systemic and local disturbances of normal growth.
