JOURNAL OF ENDODONTICS [ VOL $. NO 6, JUNE 1977
Chronic inflammation
W . G. S p e c t o r , MD, M A , L o n d o n
Chronic inflammation shares a common origin with acute inflammation in that both represent a response to a wide variety of irritants, living or dead, and that both originate in emigration of cells from the microcirculation. 1 In acute inflammation, however, the most important event is the actual transudation of leukocytes into the tissues. In chronic inflammation, the essential feature is the persistence of these leukocytes in the tissues. Thus, the exudate of acute inflammation is a loose network of fluid, fibrin, and white cells, while in chronic inflammation it is a solid accumulation of cells, most of which are of leukocytic origin. The word chronic by itself means simply long lived, but it also carries a special pathologic connotation that implies particular histologic and pathogenetic features. Many examples of acute inflammation are recurrent by nature and each episode may be marked by evidence of healing in the form of a gradually increasing degree of scarring. It has been suggested that such reactions should be termed banal chronic inflammation and that the more characteristically chronic reactions be called granulomatous. Certainly the term
granulomatous chronic inflammation has persisted and serves to delineate responses such as those of tuberculosis from the simpler tissue reaction seen, for example, in chronic cholecystitis. The word granuloma is a historic relic that we could well dispense with;
218
it would be more sensible to differentiate merely chronic from recurrent acute inflammation. Chronic inflammation would then refer to all tissue reactions in which the response was persistent and destructive or potentially destructive and in which the responding cell population was predominantly of the mononuclear phagocytic and lymphoid lines. This is the working definition implicit in the current paper. The best starting point for an understanding of chronic inflammation is the macrophage. ~ This cell forms the major constituent of most chronic inflammatory exudates, although it is augmented by neutrophilic and eosinophilic polymorphs, granulocytes, lymphocytes, plasma ceils, and fibroblasts. After many years of controversy over its origin, the macrophage is now known to be derived from a bone marrow precursormthe promonoeyte. The bone marrow cell gives rise to the monocyte, which circulates in the blood for a day or so and then enters the tissues. Most tissues of the body have their macrophage population, the cells often having a special n a m e m f o r example, Kupffer's cells in the liver and histiocytes in connective tissue. 3 The macrophage of inflammation is essentially the same as the macrophage of normal tissues, although it is likely to give evidence of heightened function. Under the light microscope, the macrophage as seen in tissue sections is a fairly nondescript mono-
nuclear cell that may be difficult to distinguish from a lymphocyte or fibroblast. With the electron microscope, the cell is seen to be highly characteristic, with a pale oval or indented nucleus, a thin rim of dense heterochromatin, a prominent nucleolus, numerous mitochondria, and lysosomes, a variable amount of rough endoplasmic reticulum, and Golgi vesicles and numerous phagocytic vacuoles. The plasma membrane is highly convoluted with many fingerlike projections. It could be deduced from these appearances that the cell is structured for phagocytosis and intracellular digestion or for secretion, or for both; this is indeed the case. A chronic inflammatory lesion may remain filled with macrophages and other cells for several years. Clearly, the body has evolved strategies for achieving this remarkable persistence, and all involved aspects of macrophage biology. The first strategy is the conversion of the macrophage to a long-lived cell that neither divides nor is replaced. It should be explained that the turnover of macrophages in the body is very variable and may be slow or fast, depending on the site and circumstances. 2 In chronic inflammation, if the macrophage phagocytizes a particle that is both indigestible and nontoxic, it sequesters it in a phagocytic vacuole and retains it for long periods--neither dividing, dying, or moving away. Chronic inflammation composed of such a stable population
JOURNAL OF ENDODONTICS I VOL 3, NO 6, JUNE 1977
of long-lived storage cells is known as a low-turnover lesion. In man, they are most likely to result from the introduction of nonirritating foreign material such as carbon. The other type of chronic inflammatory lesion, the high-turnover granuloma, is of much greater importance because it includes all the important chronic inflammatory diseases such as tuberculosis, sarcoidosis, and rheumatoid arthritis. In this variety, the cell population of the affected tissue is constantly changing. Some macrophages are dying, moving away, and draining to lymphatics while other fresh monocytes are migrating into the tissues through the vessel walls from the circulation. Once in the tissues, the macrophages undergo mitotic division and the daughter cells so provided help to maintain the size of the lesion. Monocytes migrate through vessel walls in the same way as other leukocytes by inserting pseudopodia through interendothelial junctions. They mature into macrophages by enlarging their cytoplasm and increasing the number of organelles including mitochondria, lysosomes, and endoplasmic reticulum. This is accompanied by more enzyme activity in the cell and by more membrane activity. 4 The stimulus for this sustained recruitment is twofold. There is almost certainly the local release of some chemotactic substance with a specific action on monocytes as opposed to other types of leukocytes,z Several such substances have been detected in serum, although they have not yet been purified. They may depend on activation of the complement pathway, and the trigger for their release may be in or on the macrophages already at the site of entry. Another source of monocyte chemotaxis is the sensitized lymphocyte. The daily entry of monocytes into an area of inflammation may be substantial. A small lesion provoked in the rat's foot by
injected Freund adjuvant called forth 250,000 monocytes a day for several months. 5 This influx is backed up by the appearance in the circulation of substances that promote the maturation and release of monocyte precursor cells in the bone marrow.S Mitotic division of macrophages at sites of chronic inflammation is controlled by complex factors. The cells seem impelled to divide immediately after leaving the blood vessels; after that, they remain quiescent for about two days and then may divide again. Mitosis seldom proceeds beyond approximately three generations. Division seems to be determined partly by factors in the cell and partly in the c e 11 u I a r environment. "~ Because chronic inflammation may persist for years, sustained mitotic proliferation of even a single clone of macrophages could, in theory, lead to an invasive sarcoma. It is not surprising, therefore, that cell division seems to cease after the third generation or so. Study of the chromosomes of dividing inflammatory macrophages has revealed a remarkably high proportion of defective chromosomes--gaps, breaks, and minute pairs being commonly present. From the first division, up to 25% of the cells may show these abnormalities. It seems almost certain that these abnormalities limit the viability of the ceils and restrict the number of divisions of which they are capable. A high-turnover granuloma then, which means most examples of chronic inflammation, consists of a mass of recently arrived and dividing macrophages. However, in addition to cells of a different lineage altogether, for example lymphocytes and fibrobtasts, the lesion is likely to contain two other cell types, both derNed from the macrophage. The first of these is the epithelioid cell. z This is elongated, with a very convoluted plasma membrane that interdigitates closely with
its neighbors. The cytoplasm is devoid of phagocytic vacuoles and endocytosed debris, but instead shows more rough endoplasmic reticulum and Golgi vesicles than a macrophage. Lipid droplets are present and give the cell the cloudy cytoplasm from which its name is derived. Macrophages will mature spontaneously into epithelioid cells in vivo or in vitro if they are protected from having to phagocytose poorly digestible particles. Epithelioid cells are active in pinocytosis and exocytosis but are poorly phagocytic. They have a life span of one to three weeks and are capable of mitotic division, yielding two young macrophages. They are rarely seen in low-turnover granulomas. The other macrophage derivative seen in chronic inflammation, again mainly in high-turnover lesions, is the multinucleate giant cell.~.~ This, too, is formed spontaneously in vivo and in vitro from macrophages by fusion of individual cells. Cytoplasmic bridges from two cells meet, the cell membranes fuse and disappear, and the space between the joined cytoplasmic limbs becomes a residual fusion vacuole. Many hundreds of cells may fuse in this way to form a macrophage polykaryon. Once formed, the nuclei tend to enter the mitotic cycle synchronously. If mitosis continued and proceeded to completion, gigantic structures would be formed. In fact, once the chromosomes have separated, the cycle rarely proceeds beyond metaphase. Instead, the giant cell's cytoplasm comes to contain a disorganized mass of chromosomes, some often showing premature contraction. These cells form large, bizarre mononuclear structures that are nonviable and quickly disintegrate into debris. The life span of multinucleate giant ceils is therefore quite short, seldom exceeding six days. Single macrophages in mitosis are particularly likely to be engulfed into
219
JOURNAL OF ENDODONTICS I VOL 3, NO 6, JUNE 1977
a giant cell; it seems possible that a healthy macrophage may recognize a dividing macrophage with atypical chromosomes as "foreign" and fuse with it. This is supported by experiments in which macrophages in a diffusion chamber failed to form giant ceils, although they divided freely; however, they did so when fresh cells were allowed to enter the previously sealed chamber. In any case, it seems likely that the formation of these giant cells is a useful garbage disposal system for the dividing inflammatory macrophage population, which might otherwise pose a neoplastic threat. The Langhans' giant cell appears to be merely a more organized version of the foreign-body type, because the conversion can be prevented by microtubule poisons such as colchicine. Not a great deal is known about the factors regulating these events. Macrophages divide with great reluctance when cultured in vitro but will do so if treated with cellfree inflammatory exudates, s Interestingly, however, the division is limited to one or two generations and is accompanied by chromosome defects similar to those seen in the tissues. Similarly, epithelioid change and giant cell formation occur much less readily in vitro than in vivo, and the latter at least can be accelerated by cellfree inflammatory exudate. Having analyzed the strategy by which macrophage infiltration persists in chronic inflammation, it remains to discuss the pathological mechanisms that induce the persistence. Broadly speaking, there are only three reasons for chronic inflammation to persist: retention of extracellular irritant at the site of injury; persistence of irritant within the cytoplasm of macrophages; and instruction from sensitized lymphocytes. Needless to say, combinations of all three may occur. Of
220
these mechanisms, extracellular persistence is the least important. Deposits such as metal or large masses of inorganic material are more likely to lead to a quiescent lesion or to an inert fibrous capsule. Where active inflammation exists in such cases, for example in silicosis, the extracellular masses are unimportant compared with the intracellular accumulation within macrophages. The importance of intracellular persistence in macrophages is easily demonstrated by injecting radioactivelabeled particles such as bacteria into the tissues. 9 As long as radioactivity is detectable inside the cytoplasm of the locally infiltrating macrophages, the chronic inflammation will persist and vice versa. However, when one disappears, so does the other. If the bacteria were not labeled, it would have been very difficult to discover their presence and the granuloma would have appeared to have no obvious reason for its existence. By no means do all injected particles cause persistent macrophage accumulation in the tissues. Staphylococcus albus is rapidly eliminated, while Bordetella pertussis and Mycobacterium tuberculosis remain. Streptococcus pyogenes is rapidly digested, but the L form of the same organism is retained intracellularly for weeks. Soluble antigen-antibody complexes prepared in antigen excess disappear rapidly, but insoluble complexes of the same antigen prepared in antibody excess remain in macrophages for weeks. 1~ It is obvious that failure of macrophages to eliminate the material they phagocytose is a vital factor in perpetuating an inflammatory reaction. There are several reasons for such failure. In the first instance, bacteria may be ingested and survive and even multiply inside the macrophage's cytoplasm as happens in tuberculosis and leprosy. It is far from clear how macrophages normally kill ingested
microorganisms. The reactions involved may include digestion of the bacterial cell wall by lysozymes or the bactericidal effect of hydrogen peroxide with or without catalase or the formation of bactericidal aldehydes, following peroxidation of intracellular lipids. The battery is more limited than in the case of granulocytes, which contain the powerful myeloperoxidase-halide system. It is not surprising, therefore, that certain complex bacteria or fungi can survive inside macrophages. Viruses no doubt survive inside the cells by their wellknown strategies such as formation of provirus. Quite apart from the problem of bacterial survival is the persistence of dead bacteria, of bacterial fragments, or of nonliving matter. 11 The problem in this case is that the cell is unable to digest the ingested residue to its soluble breakdown products. Because culturing of many granulomas yields no living organisms, this mechanism can be regarded as highly important. Once again, there are several reasons why intracellular material might fail to be digested. The normal process involves entry of the material into a phagocytic vacuole, fusion of lysosomes with the phagocytic vacuole to form a phagosome, entry of lysosomal digestive enzymes into the phagosome, and, finally, digestion of the particle to soluble components that diffuse out of the cell. The process may fail at the first step if the material is not ingested. This occurs in the case of certain worm larvae that become coated with host antigen and are thus recognized wrongly as "self. ''12 There m a y be failure of the lysosomes to fuse with the phagocytic vacuole. This happens in tuberculosis and may be caused by secretion by the mycobacteria of an inhibitor of the fusion process. The enzymes m a y enter the phagosome but be unable to attack the ingested particle if the particular
JOURNAL OF ENDODONTICS I VOL 3, NO 6, JUNE 1977
enzyme required is absent. This probably happens when very foreign material, for example seaweed extract, is endocytosed. More important, the lysosomal enzymes may partially digest the bacteria until a structure is formed that they cannot attack. The structure seems especially likely to be the bacterial cell wall or even quite simple molecules (peptidoglycans) derived from them. Another possibility is that substances in the cellular environment may prevent digestion. This property is possessed by certain anionic and cationic polyelectrolytes, which may be formed by degradation of connective tissue and which seem to act as inhibitors of the lysosomal enzymes. Even specific antibody may delay digestion of dead bacteria for reasons not yet clear. To summarize this complex subject, particulate matter may remain undigested in the macrophage's cytoplasm and thereby cause chronic inflammation because the proper enzymes are lacking, the material is inherently nonbiodegradable, or the factors in the particle or environment prevent enzyme action. The other major mechanism for causing persistent inflammatory infiltration is the sensitized lymphocyte, that is, cell-mediated immunity. 13 The best example of this is schistosomiasis, in which the larvae produce virtually no cellular reaction unless T-cellmediated hypersensitivity is present. 14 Zirconium and other rare metals may produce similar granulomatous reactions caused by cell-mediated immunity in man, although in some idiosyncratic individuals only. Sensitized lymphocytes may well aggravate chronic inflammation even when a particulate irritant is plentiful, as in tuberculosis. On the other hand, neonatal thymectomy, which depletes the body of T cells, has little effect on many inflammatory granulomas. The mechanism involved in lymphocyte-macrophage
interaction is probably the release from lymphocytes of a family of substances called lymphokines. 15 These have many actions, including the chemotaxis of macrophages enhancing their bactericidal powers and also of immobilizing macrophages. They include the mysterious "transfer factor," which restores cellular immunity when it is deficient. The foregoing presents a reasonable explanation of the anatomy of chronic inflammation, of the ways in which chronicity is maintained, and why it occurs. Chronic inflammation is, however, not merely accumulative but also destructive of existing tissues. The erosion of articular cartilage in rheumatoid arthritis and the crippling fibrosis of silicosis are examples of this effect. It has already been mentioned that the macrophage not only releases lysosomal enzymes, but continues to secrete and excrete them for sustained periods. 15 These enzymes, which digest so much endocytosed material, are highly destructive if sent into the cell's environment. They contain enzymes capable of attacking proteins, carbohydrates, and lipids, and of functioning at different levels of tissue acidity and ionic strength. They are easily identified in the areas where they are doing mischief, as in the synovial fluid of rheumatoid joints. The secretion from macrophages is augmented by the uptake of particulate matter, especially if it is an irritant such as silica; secretion may continue as long as the irritant remains. Phagocytosis also promotes the release of a factor that causes fibroblasts to divide and to synthesize collagen and ground substance. Thus, persistence of intracetlular irritant explains not only the recalcitrant cellular infiltration of chronic inflammation, but also the destructive effects of this exudate. It is likely, however, that lymphocytes also can instruct macrophages to secrete these erosive
products. In other words, tissue destruction in chronic inflammation can be either a simple cell response to uptake of an irritant or be dependent on complex immunologic mechanisms. To those familiar with the problems of periodontal disease, the significance of many of the aforementioned observations is apparent. Equally apparent, however, is the need for further work to clarify which particular effects are most likely to be of relevance. Dr. Spector is a professor, department of pathology, St. Bartholomew's Hospital Medical College, London. Requests for reprints should be directed to Dr. W. G. Spector. References
1. Spector, W.G. The granulomatous inflammatory exudate. Int Rev Exp Pathol 8:1, 1969. 2. Spector, W.G. The macrophage: its origins and role in pathology. Pathobiol Annu 4:33, 1974. 3. Van Furth, R.; Cohn, Z.A.; Hirsch, J.G.; Humphrey, J.H.; Spector, W.G.; and Langevoort, H.L. The mononuclear phagocyte system; a new classification of macrophages, monocytes and their precursor cells. Bull WHO 46:845, 1972. 4. Karnovsky, M.L.; Lasdius, J.; and Simmons, S.R. Metabolism of activated mononuclear phagocytes at rest and during phagocytosis. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 423. 5. Spector, W.G.; Lykke, A.W.J.; and Willoughby, D.A. A quantitative study of leucocyte emigration in chronic inflammatory granulomata. J Pathol Bacteriol 93:101 Jan 1967. 6. Mariano, M., and Spector, W.G. The formation and properties of macrophage polykaryons (inflammatory giant cells). J Pathol 113:1 May 1974. 7. Wynne, K.M.; Spectro, W.G.; and Willoughby, D.A. Macrophage proliferation in vitro induced by exudates. Nature 253:636 Feb 1975. 8. Spector, W.G.; Reichold, N.; and Ryan, G.B. Degradation of granulomainducing microorganisms by macrophages. J Pathol 101:339 Aug 1970.
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JOURNAL OF ENDODONTICS I VOL 3, NO 6, JUNE 1977
9. Spector, W.G., and Heesom, N. The production of granulomata by antigen/antibody complexes. J Pathol 98:31 May 1969. 10. Klebanoff, S.J., and Hammon, C.B. Antimicrobial systems of mononuclear phagocytes. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 507. 11. Smithers, S.R.; Terry, R.J.; and Hockley, D.J. Host antigens in schistosomiasis. Proc R Soc B 171:483 Feb 1969. 12. Draper, P., and D'arcy, H.P. Phagosomes, lysosomes and mycobacteria:
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cellular and microbial aspects. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 575. 13. Warren, K.S., and Boros, D.L. The schistosome egg granuloma--a form of cell mediated immunity. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 1015. 14. Dumonde, D.C.; Kelly, R.H.; Preston, P.M.; and Wolstencroft, R.A. Lymphokines and macrophage function in the
immunological response. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 675. 15. Allison, A.C., and Davies, P. In search of biochemical and biological activities of mononuclear phagocytes exposed to various stimuli, with special reference to secretion of lysosomal enzymes. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 487.
Chronic inflammation
W . G. S p e c t o r , MD, M A , L o n d o n
Chronic inflammation shares a common origin with acute inflammation in that both represent a response to a wide variety of irritants, living or dead, and that both originate in emigration of cells from the microcirculation. 1 In acute inflammation, however, the most important event is the actual transudation of leukocytes into the tissues. In chronic inflammation, the essential feature is the persistence of these leukocytes in the tissues. Thus, the exudate of acute inflammation is a loose network of fluid, fibrin, and white cells, while in chronic inflammation it is a solid accumulation of cells, most of which are of leukocytic origin. The word chronic by itself means simply long lived, but it also carries a special pathologic connotation that implies particular histologic and pathogenetic features. Many examples of acute inflammation are recurrent by nature and each episode may be marked by evidence of healing in the form of a gradually increasing degree of scarring. It has been suggested that such reactions should be termed banal chronic inflammation and that the more characteristically chronic reactions be called granulomatous. Certainly the term
granulomatous chronic inflammation has persisted and serves to delineate responses such as those of tuberculosis from the simpler tissue reaction seen, for example, in chronic cholecystitis. The word granuloma is a historic relic that we could well dispense with;
218
it would be more sensible to differentiate merely chronic from recurrent acute inflammation. Chronic inflammation would then refer to all tissue reactions in which the response was persistent and destructive or potentially destructive and in which the responding cell population was predominantly of the mononuclear phagocytic and lymphoid lines. This is the working definition implicit in the current paper. The best starting point for an understanding of chronic inflammation is the macrophage. ~ This cell forms the major constituent of most chronic inflammatory exudates, although it is augmented by neutrophilic and eosinophilic polymorphs, granulocytes, lymphocytes, plasma ceils, and fibroblasts. After many years of controversy over its origin, the macrophage is now known to be derived from a bone marrow precursormthe promonoeyte. The bone marrow cell gives rise to the monocyte, which circulates in the blood for a day or so and then enters the tissues. Most tissues of the body have their macrophage population, the cells often having a special n a m e m f o r example, Kupffer's cells in the liver and histiocytes in connective tissue. 3 The macrophage of inflammation is essentially the same as the macrophage of normal tissues, although it is likely to give evidence of heightened function. Under the light microscope, the macrophage as seen in tissue sections is a fairly nondescript mono-
nuclear cell that may be difficult to distinguish from a lymphocyte or fibroblast. With the electron microscope, the cell is seen to be highly characteristic, with a pale oval or indented nucleus, a thin rim of dense heterochromatin, a prominent nucleolus, numerous mitochondria, and lysosomes, a variable amount of rough endoplasmic reticulum, and Golgi vesicles and numerous phagocytic vacuoles. The plasma membrane is highly convoluted with many fingerlike projections. It could be deduced from these appearances that the cell is structured for phagocytosis and intracellular digestion or for secretion, or for both; this is indeed the case. A chronic inflammatory lesion may remain filled with macrophages and other cells for several years. Clearly, the body has evolved strategies for achieving this remarkable persistence, and all involved aspects of macrophage biology. The first strategy is the conversion of the macrophage to a long-lived cell that neither divides nor is replaced. It should be explained that the turnover of macrophages in the body is very variable and may be slow or fast, depending on the site and circumstances. 2 In chronic inflammation, if the macrophage phagocytizes a particle that is both indigestible and nontoxic, it sequesters it in a phagocytic vacuole and retains it for long periods--neither dividing, dying, or moving away. Chronic inflammation composed of such a stable population
JOURNAL OF ENDODONTICS I VOL 3, NO 6, JUNE 1977
of long-lived storage cells is known as a low-turnover lesion. In man, they are most likely to result from the introduction of nonirritating foreign material such as carbon. The other type of chronic inflammatory lesion, the high-turnover granuloma, is of much greater importance because it includes all the important chronic inflammatory diseases such as tuberculosis, sarcoidosis, and rheumatoid arthritis. In this variety, the cell population of the affected tissue is constantly changing. Some macrophages are dying, moving away, and draining to lymphatics while other fresh monocytes are migrating into the tissues through the vessel walls from the circulation. Once in the tissues, the macrophages undergo mitotic division and the daughter cells so provided help to maintain the size of the lesion. Monocytes migrate through vessel walls in the same way as other leukocytes by inserting pseudopodia through interendothelial junctions. They mature into macrophages by enlarging their cytoplasm and increasing the number of organelles including mitochondria, lysosomes, and endoplasmic reticulum. This is accompanied by more enzyme activity in the cell and by more membrane activity. 4 The stimulus for this sustained recruitment is twofold. There is almost certainly the local release of some chemotactic substance with a specific action on monocytes as opposed to other types of leukocytes,z Several such substances have been detected in serum, although they have not yet been purified. They may depend on activation of the complement pathway, and the trigger for their release may be in or on the macrophages already at the site of entry. Another source of monocyte chemotaxis is the sensitized lymphocyte. The daily entry of monocytes into an area of inflammation may be substantial. A small lesion provoked in the rat's foot by
injected Freund adjuvant called forth 250,000 monocytes a day for several months. 5 This influx is backed up by the appearance in the circulation of substances that promote the maturation and release of monocyte precursor cells in the bone marrow.S Mitotic division of macrophages at sites of chronic inflammation is controlled by complex factors. The cells seem impelled to divide immediately after leaving the blood vessels; after that, they remain quiescent for about two days and then may divide again. Mitosis seldom proceeds beyond approximately three generations. Division seems to be determined partly by factors in the cell and partly in the c e 11 u I a r environment. "~ Because chronic inflammation may persist for years, sustained mitotic proliferation of even a single clone of macrophages could, in theory, lead to an invasive sarcoma. It is not surprising, therefore, that cell division seems to cease after the third generation or so. Study of the chromosomes of dividing inflammatory macrophages has revealed a remarkably high proportion of defective chromosomes--gaps, breaks, and minute pairs being commonly present. From the first division, up to 25% of the cells may show these abnormalities. It seems almost certain that these abnormalities limit the viability of the ceils and restrict the number of divisions of which they are capable. A high-turnover granuloma then, which means most examples of chronic inflammation, consists of a mass of recently arrived and dividing macrophages. However, in addition to cells of a different lineage altogether, for example lymphocytes and fibrobtasts, the lesion is likely to contain two other cell types, both derNed from the macrophage. The first of these is the epithelioid cell. z This is elongated, with a very convoluted plasma membrane that interdigitates closely with
its neighbors. The cytoplasm is devoid of phagocytic vacuoles and endocytosed debris, but instead shows more rough endoplasmic reticulum and Golgi vesicles than a macrophage. Lipid droplets are present and give the cell the cloudy cytoplasm from which its name is derived. Macrophages will mature spontaneously into epithelioid cells in vivo or in vitro if they are protected from having to phagocytose poorly digestible particles. Epithelioid cells are active in pinocytosis and exocytosis but are poorly phagocytic. They have a life span of one to three weeks and are capable of mitotic division, yielding two young macrophages. They are rarely seen in low-turnover granulomas. The other macrophage derivative seen in chronic inflammation, again mainly in high-turnover lesions, is the multinucleate giant cell.~.~ This, too, is formed spontaneously in vivo and in vitro from macrophages by fusion of individual cells. Cytoplasmic bridges from two cells meet, the cell membranes fuse and disappear, and the space between the joined cytoplasmic limbs becomes a residual fusion vacuole. Many hundreds of cells may fuse in this way to form a macrophage polykaryon. Once formed, the nuclei tend to enter the mitotic cycle synchronously. If mitosis continued and proceeded to completion, gigantic structures would be formed. In fact, once the chromosomes have separated, the cycle rarely proceeds beyond metaphase. Instead, the giant cell's cytoplasm comes to contain a disorganized mass of chromosomes, some often showing premature contraction. These cells form large, bizarre mononuclear structures that are nonviable and quickly disintegrate into debris. The life span of multinucleate giant ceils is therefore quite short, seldom exceeding six days. Single macrophages in mitosis are particularly likely to be engulfed into
219
JOURNAL OF ENDODONTICS I VOL 3, NO 6, JUNE 1977
a giant cell; it seems possible that a healthy macrophage may recognize a dividing macrophage with atypical chromosomes as "foreign" and fuse with it. This is supported by experiments in which macrophages in a diffusion chamber failed to form giant ceils, although they divided freely; however, they did so when fresh cells were allowed to enter the previously sealed chamber. In any case, it seems likely that the formation of these giant cells is a useful garbage disposal system for the dividing inflammatory macrophage population, which might otherwise pose a neoplastic threat. The Langhans' giant cell appears to be merely a more organized version of the foreign-body type, because the conversion can be prevented by microtubule poisons such as colchicine. Not a great deal is known about the factors regulating these events. Macrophages divide with great reluctance when cultured in vitro but will do so if treated with cellfree inflammatory exudates, s Interestingly, however, the division is limited to one or two generations and is accompanied by chromosome defects similar to those seen in the tissues. Similarly, epithelioid change and giant cell formation occur much less readily in vitro than in vivo, and the latter at least can be accelerated by cellfree inflammatory exudate. Having analyzed the strategy by which macrophage infiltration persists in chronic inflammation, it remains to discuss the pathological mechanisms that induce the persistence. Broadly speaking, there are only three reasons for chronic inflammation to persist: retention of extracellular irritant at the site of injury; persistence of irritant within the cytoplasm of macrophages; and instruction from sensitized lymphocytes. Needless to say, combinations of all three may occur. Of
220
these mechanisms, extracellular persistence is the least important. Deposits such as metal or large masses of inorganic material are more likely to lead to a quiescent lesion or to an inert fibrous capsule. Where active inflammation exists in such cases, for example in silicosis, the extracellular masses are unimportant compared with the intracellular accumulation within macrophages. The importance of intracellular persistence in macrophages is easily demonstrated by injecting radioactivelabeled particles such as bacteria into the tissues. 9 As long as radioactivity is detectable inside the cytoplasm of the locally infiltrating macrophages, the chronic inflammation will persist and vice versa. However, when one disappears, so does the other. If the bacteria were not labeled, it would have been very difficult to discover their presence and the granuloma would have appeared to have no obvious reason for its existence. By no means do all injected particles cause persistent macrophage accumulation in the tissues. Staphylococcus albus is rapidly eliminated, while Bordetella pertussis and Mycobacterium tuberculosis remain. Streptococcus pyogenes is rapidly digested, but the L form of the same organism is retained intracellularly for weeks. Soluble antigen-antibody complexes prepared in antigen excess disappear rapidly, but insoluble complexes of the same antigen prepared in antibody excess remain in macrophages for weeks. 1~ It is obvious that failure of macrophages to eliminate the material they phagocytose is a vital factor in perpetuating an inflammatory reaction. There are several reasons for such failure. In the first instance, bacteria may be ingested and survive and even multiply inside the macrophage's cytoplasm as happens in tuberculosis and leprosy. It is far from clear how macrophages normally kill ingested
microorganisms. The reactions involved may include digestion of the bacterial cell wall by lysozymes or the bactericidal effect of hydrogen peroxide with or without catalase or the formation of bactericidal aldehydes, following peroxidation of intracellular lipids. The battery is more limited than in the case of granulocytes, which contain the powerful myeloperoxidase-halide system. It is not surprising, therefore, that certain complex bacteria or fungi can survive inside macrophages. Viruses no doubt survive inside the cells by their wellknown strategies such as formation of provirus. Quite apart from the problem of bacterial survival is the persistence of dead bacteria, of bacterial fragments, or of nonliving matter. 11 The problem in this case is that the cell is unable to digest the ingested residue to its soluble breakdown products. Because culturing of many granulomas yields no living organisms, this mechanism can be regarded as highly important. Once again, there are several reasons why intracellular material might fail to be digested. The normal process involves entry of the material into a phagocytic vacuole, fusion of lysosomes with the phagocytic vacuole to form a phagosome, entry of lysosomal digestive enzymes into the phagosome, and, finally, digestion of the particle to soluble components that diffuse out of the cell. The process may fail at the first step if the material is not ingested. This occurs in the case of certain worm larvae that become coated with host antigen and are thus recognized wrongly as "self. ''12 There m a y be failure of the lysosomes to fuse with the phagocytic vacuole. This happens in tuberculosis and may be caused by secretion by the mycobacteria of an inhibitor of the fusion process. The enzymes m a y enter the phagosome but be unable to attack the ingested particle if the particular
JOURNAL OF ENDODONTICS I VOL 3, NO 6, JUNE 1977
enzyme required is absent. This probably happens when very foreign material, for example seaweed extract, is endocytosed. More important, the lysosomal enzymes may partially digest the bacteria until a structure is formed that they cannot attack. The structure seems especially likely to be the bacterial cell wall or even quite simple molecules (peptidoglycans) derived from them. Another possibility is that substances in the cellular environment may prevent digestion. This property is possessed by certain anionic and cationic polyelectrolytes, which may be formed by degradation of connective tissue and which seem to act as inhibitors of the lysosomal enzymes. Even specific antibody may delay digestion of dead bacteria for reasons not yet clear. To summarize this complex subject, particulate matter may remain undigested in the macrophage's cytoplasm and thereby cause chronic inflammation because the proper enzymes are lacking, the material is inherently nonbiodegradable, or the factors in the particle or environment prevent enzyme action. The other major mechanism for causing persistent inflammatory infiltration is the sensitized lymphocyte, that is, cell-mediated immunity. 13 The best example of this is schistosomiasis, in which the larvae produce virtually no cellular reaction unless T-cellmediated hypersensitivity is present. 14 Zirconium and other rare metals may produce similar granulomatous reactions caused by cell-mediated immunity in man, although in some idiosyncratic individuals only. Sensitized lymphocytes may well aggravate chronic inflammation even when a particulate irritant is plentiful, as in tuberculosis. On the other hand, neonatal thymectomy, which depletes the body of T cells, has little effect on many inflammatory granulomas. The mechanism involved in lymphocyte-macrophage
interaction is probably the release from lymphocytes of a family of substances called lymphokines. 15 These have many actions, including the chemotaxis of macrophages enhancing their bactericidal powers and also of immobilizing macrophages. They include the mysterious "transfer factor," which restores cellular immunity when it is deficient. The foregoing presents a reasonable explanation of the anatomy of chronic inflammation, of the ways in which chronicity is maintained, and why it occurs. Chronic inflammation is, however, not merely accumulative but also destructive of existing tissues. The erosion of articular cartilage in rheumatoid arthritis and the crippling fibrosis of silicosis are examples of this effect. It has already been mentioned that the macrophage not only releases lysosomal enzymes, but continues to secrete and excrete them for sustained periods. 15 These enzymes, which digest so much endocytosed material, are highly destructive if sent into the cell's environment. They contain enzymes capable of attacking proteins, carbohydrates, and lipids, and of functioning at different levels of tissue acidity and ionic strength. They are easily identified in the areas where they are doing mischief, as in the synovial fluid of rheumatoid joints. The secretion from macrophages is augmented by the uptake of particulate matter, especially if it is an irritant such as silica; secretion may continue as long as the irritant remains. Phagocytosis also promotes the release of a factor that causes fibroblasts to divide and to synthesize collagen and ground substance. Thus, persistence of intracetlular irritant explains not only the recalcitrant cellular infiltration of chronic inflammation, but also the destructive effects of this exudate. It is likely, however, that lymphocytes also can instruct macrophages to secrete these erosive
products. In other words, tissue destruction in chronic inflammation can be either a simple cell response to uptake of an irritant or be dependent on complex immunologic mechanisms. To those familiar with the problems of periodontal disease, the significance of many of the aforementioned observations is apparent. Equally apparent, however, is the need for further work to clarify which particular effects are most likely to be of relevance. Dr. Spector is a professor, department of pathology, St. Bartholomew's Hospital Medical College, London. Requests for reprints should be directed to Dr. W. G. Spector. References
1. Spector, W.G. The granulomatous inflammatory exudate. Int Rev Exp Pathol 8:1, 1969. 2. Spector, W.G. The macrophage: its origins and role in pathology. Pathobiol Annu 4:33, 1974. 3. Van Furth, R.; Cohn, Z.A.; Hirsch, J.G.; Humphrey, J.H.; Spector, W.G.; and Langevoort, H.L. The mononuclear phagocyte system; a new classification of macrophages, monocytes and their precursor cells. Bull WHO 46:845, 1972. 4. Karnovsky, M.L.; Lasdius, J.; and Simmons, S.R. Metabolism of activated mononuclear phagocytes at rest and during phagocytosis. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 423. 5. Spector, W.G.; Lykke, A.W.J.; and Willoughby, D.A. A quantitative study of leucocyte emigration in chronic inflammatory granulomata. J Pathol Bacteriol 93:101 Jan 1967. 6. Mariano, M., and Spector, W.G. The formation and properties of macrophage polykaryons (inflammatory giant cells). J Pathol 113:1 May 1974. 7. Wynne, K.M.; Spectro, W.G.; and Willoughby, D.A. Macrophage proliferation in vitro induced by exudates. Nature 253:636 Feb 1975. 8. Spector, W.G.; Reichold, N.; and Ryan, G.B. Degradation of granulomainducing microorganisms by macrophages. J Pathol 101:339 Aug 1970.
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9. Spector, W.G., and Heesom, N. The production of granulomata by antigen/antibody complexes. J Pathol 98:31 May 1969. 10. Klebanoff, S.J., and Hammon, C.B. Antimicrobial systems of mononuclear phagocytes. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 507. 11. Smithers, S.R.; Terry, R.J.; and Hockley, D.J. Host antigens in schistosomiasis. Proc R Soc B 171:483 Feb 1969. 12. Draper, P., and D'arcy, H.P. Phagosomes, lysosomes and mycobacteria:
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cellular and microbial aspects. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 575. 13. Warren, K.S., and Boros, D.L. The schistosome egg granuloma--a form of cell mediated immunity. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 1015. 14. Dumonde, D.C.; Kelly, R.H.; Preston, P.M.; and Wolstencroft, R.A. Lymphokines and macrophage function in the
immunological response. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 675. 15. Allison, A.C., and Davies, P. In search of biochemical and biological activities of mononuclear phagocytes exposed to various stimuli, with special reference to secretion of lysosomal enzymes. In Van Furth, R., ed. Mononuclear phagocytes in infection, immunity and pathology. Oxford, England, Blackwell Scientific Publications, 1975, p 487.
