Aspects of cell biology of the normal periodontium

Periodontology 2000, Val. 3. 1993, 64-75 Printed in Denmark . All righrs resented

Copyright 0 Munksgaard 1993

PERIODONTOLOGY 2000 ISSN 0906-6713

Aspects of cell biology normal periodontium ARTHUR F. HEFTI

In this volume, Thomas M. Hassell has provided a detailed description of the tissues and cells of the healthy periodontium from an anatomic and histo- logical perspective. Angelo Mariotti has offered the reader a comprehensive exposure to the structures of the macromolecular components of healthy periodontal tissues. A principal construct of periodontal normalcy has emerged; however, there is a danger that this attractive construct could be interpreted as a static situation of health that remains unchanged and unchanging until some insult initiates incipient pathological alteration. On the contrary, the periodontal tissues are among the most biologically active in the body, exhibiting a remarkably high turnover rate among the cells and tissues. Health (normalcy) in the periodontium is the result and sum of an ongoing dynamic characterized by both anabolic and catabolic activities. Tissue homeostasis represents a delicate balance between these two antagonistic processes. The production and the de- struction of tissue matrix (turnover) in a healthy state involves interaction among a myriad of effector molecules that are synthesized and secreted by the resident cells themselves. These effector molecules have been investigated extensively in recent years, interestingly enough, primarily in studies of periodontal diseases (31). Growth factors and cytokines that are believed to play primary roles in the pathogenesis of gingivitis and periodontitis are probably the same ones that operate to maintain periodontal homeostasis in health. The balance between effector molecule-induced tissue breakdown and tissue formation is the essence of periodontal health.

Soft connective tissue is the predominant com- ponent of the gingiva and the periodontal ligament. Its most important components are collagen fibers, fibroblasts and interstitial tissue matrix elements. A fundamental question in periodontal biology is how these components interact to maintain the contour, integrity and function of the healthy tissue. In other words, what is the molecular basis for normal con-

of the

nective tissue remodeling and turnover? There is abundant evidence that cytokines, which are secreted by fibroblasts (641, endothelial cells (751, epithelial cells and cells of the immune system (91, play a decisive role in tissue homeostasis, acting in con- cert with matrix metalloproteinases and their natural inhibitors, tissue inhibitors of metalloproteinases. This chapter describes some of the most important regulatory molecules found in connective tissue and outlines how such molecules could affect connective tissue matrix turnover in health.

Cytokines

Early in uitro studies of cell proliferation suggested that embryonic fluid may contain substances that promote growth activity. When Carrel (13) exposed chicken fibroblasts to an embryonic extract, he observed enhanced cell proliferation; consequently, he suggested that such substances may be important during tissue repair or tissue regeneration. It was not until 40 years later, however, that Kasakura & Low- enstein (49) demonstrated that cell-free soluble factors generated in uitro in the culture supernatants of sensitized white blood cells incubated with antigen could mimic delayed-type hypersensitivity, and that they were mitogenic for lymphocytes. Such cell-free soluble factors were termed lymphokines. Subse- quently, over 100 lymphokine activities were described (go), but only by the late 1970s did technical progress lead to the biochemical purification of a few lymphokines. It is now clear that the great variety of observations made in experiments using cell culture supernatants were actually the result of a small number of molecules with multiple, overlap- ping biological activities. For example, the factor derived from monocytes, which caused lymphocyte activation, appeared to be chemically identical to the activity that previously was known as B-cell activating factor or mononuclear cell factor. In 1978, the

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Aspects of cell biology of the normal periodontiurn

term interleukin- 1 was proposed to substitute for lymphocyte activation factor, B-cell activating factor and mononuclear cell factor. Similarly, the name interleukin-2 would replace T-cell growth factor, lymphocyte mitogenic factor and blastogenic factor (61). The term cytokine, initially proposed by Cohen et al. (171, describes a series of multifunctional polypeptides and glycoproteins that are secreted by one or several cell types and act locally or systemically. Included in the cytokine molecule group are interleukins, cytotoxic factors, interferons, growth factors, colony-stimulating factors and intercrines (42).

A comprehensive list of human cytokines, their molecular weights and cell sources is reproduced in Table 1.

What immunologists call interleukins, cell biol- ogists have sometimes called growth factors. Growth factors have been defined as substances capable of re-initiating proliferation of cells that are in a quies- cent state (that is, competence growth factor). Once cells enter the G, phase of cell cycle, other factors (progression growth factors) are needed for tran- sition into the S phase and subsequent cell division. However, the term growth factor may be somewhat

Table 1. The human cvtokines. AdaDted and modified from Burke et al. (9) and Henderson & Blake (42) Molecular weight Amino acid

Cytokine or growth factor (kDa) content Major cell source Interleukins Interleukin-la

Interleukin- l p Interleukin-2 Interleukin-3 Interleukin-4 Interleukin-5 Interleukin-6

Interleukin-7 Interleukin-8

Interleukin-9 Interleukin-10 (murine) Interleukin-1 1 Interleukin- 12

Cytotoxic factors Tumor necrosis factor-a

Tumor necrosis factor-p Interferons Interferon-a Interferon$ Interferon- y

Growth factors Epidermal growth factor Acidic fibroblast growth factor Basic fibroblast growth factor Insulin-like growth factor-1 Platelet-derived growth factor- 1

Transforming growth factor-a Transforming growth factor-p

Colony-stimulating factors Granulocyte colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor

17.5

17.5 15-20 14-30 15-19

2X21.5 21-28

25 6-10

32-39 19 23 70

17

25

16-27 20

20-25

6 15-18 15-18

7 28-35

5-6 25

20

22

159

153 133 133 129

2x113 184

152 72

179 160 199

157

171

166 166 146

53 149 146 70

2x104

50 2x112

177

127

224

monocyte/macrophage, endothelial cell, fibroblast keratinocyte T-cell T-cell, mast cell T-cell, mast cell T-cell, mast cell macrophage, fibroblast, T-cell, endothelial cell bone marrow cells monocyte, fibroblast, endothelial cell, keratinocyte T-cell T-cell bone marrow cell B-cell

monocyte/macrophage, fibroblast, T-cell T-cell, B-cell

macrophage fibroblast T-cell, natural killer cell

macrophage platelet, macrophage endothelial cell macro phage platelet, monocyte/macrophage, endothelial cell macrophage platelet, monocyte/macrophage, T-cell, fibroblast, endothelial cell

monocytelmacrophage, fibroblast, endothelial cell T-cell, fibroblast, endothelial cell

fibroblast. monocvte. endothelial cell Macrophage colonykmulating factor 70-90 , I

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misleading, since it is known that these compounds can display both stimulatory and inhibitory activities in vitro, even with the same cell type (72). The re- sulting cell response may then depend on the presence of other cytolunes, the state of cell activation and the degree of cell differentiation. In vivo, cytokines are believed to play an important role in numerous biological events, including development, homeostasis, regeneration, repair, inflammation and neoplasia. The following section presents a selection of cytokines that are deemed important with regard to tissue homeostasis. The overview, however, is brief and has to be incomplete. For more comprehensive coverage of the subject, see the specific literature.

Fibroblast growth factor

A well characterized example of a cytokine family are the fibroblast growth factors, which can be found in many tissues. Two of 7 isoforms of fibroblast growth factor have been isolated and described in particular detail; one is basic and the other acidic. Basic fibroblast growth factor was discovered by its ability to induce proliferation in mouse fibroblasts; acidic fibroblast growth factor was found to delay differentiation of myoblasts and to stimulate endothelial cell proliferation. The two fibroblast growth factors are products of different genes (46, 60, 75) but are similar in structure and biological activities. Both fibroblast growth factors are single-chain proteins with molecular weights ranging from 15 to 18 kDa and share 53% homology in their amino acid sequences (27). The human acidic fibroblast growth factor and basic fibroblast growth factor genes are single-copy genes located on chromosomes 5 and 4, respectively. Their expression appears to be independently regulated. The fibroblast growth factor mRNA levels in cells are normally very low, possibly because of

mRNA instability, but tissue levels are relatively high. The predominant view is that fibroblast growth factors are not humoral factors but rather locally active tissue factors (85) integrated within the base- ment membrane.

Acidic and basic fibroblast growth factors interact with the same membrane receptors (see Walters in this volume). Basic fibroblast growth factor has a higher affinity than acidic fibroblast growth factor for the 145 kDa receptor species, whereas acidic fibroblast growth factor exhibits higher affinity for the 125 kDa receptor. The highly homologous amino acid compositions of the two fibroblast growth factors suggest an interaction with their surface receptors through similar binding domains (66).

Acidic fibroblast growth factor is primarily known for its effects on endothelial cell replication and neo- vascularization. In bone tissue cultures, acidic fibroblast growth factor stimulates DNA synthesis and cell replication, which results in increased protein synthesis (ll), especially of collagen type I. Like acidic fibroblast growth factor, basic fibroblast growth factor has angiogenic properties and is highly chemotactic and mitogenic for a variety of cell types (Tables 2-4). Similarly, it stimulates bone cell replication and increases the number of cells of the osteoblastic lineage. Fibroblast growth factors bind to heparin sulfate, heparin and fibronectin in the extracellular matrix. Heparin binding may be an especially important property of acidic fibroblast growth factor, since it has been shown (32) that the combination of heparin plus acidic fibroblast growth factor increases the mitogenic activity of acidic fibroblast growth factor on BALB/C3T3 fibroblasts by greater than 100-fold. In contrast, heparin does not enhance the activity of basic fibroblast growth factor. Thus, in the presence of heparin, the 2 forms of fibroblast growth factor are essentially equipo- tent. Heparin protects acidic fibroblast growth factor from inactivation, but one of the major differences

Table 2. Effect of selected cytokines on cell migration by various cell types. PDGF: platelet derived growth factor; TGF-alp: transforming growth factor-alp; EGF: epidermal growth factor; FGF: fibroblast growth factor. -: inhibition; 0: no effect; +: slight effect; + +: moderate effect; + + +: strong effect

Epithelial cells Fibroblasts Endothelial cells Inflammatory cells Osteoblasts

PDGF 0 -+++I 0 01+ (1 TGF-cx F l + + 0 ?

TGF-P + + ? /TTl ?

FGF Fl -1 -1 0 ?

EGF + 0 0 ?

Adapted from Lynch & Giannobile (57) with permission.

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Table 3. Effect of selected cytokines on mitogenesis of various cell types. PDGF: platelet-derived growth factor; TGF-alp: transforming growth factor-alp; EGF: epidermal growth factor; FGF: fibroblast growth factor. -: inhibition; 0: no effect: +: slight effect; ++: moderate effect; +++: strong effect

~~ ~~ ~

Epithelial cells Fibroblasts Endothelial cells Smooth muscle Osteoblasts cells

PDGF 0 p q 0 p 7 - l F] TGF-a FJ + + ? +

? + - +I- TGF-D -

EGF I +++ I + + ?

FGF pq ++ Vl + + +

Adapted from Lynch & Giannobile (57) with permission.

between the actions of acidic fibroblast growth factor and basic fibroblast growth factor is that the effect of basic fibroblast growth factor on cell replication in some cell cultures is not increased by heparin, as is that of acidic fibroblast growth factor. Fibroblast growth factor is a potent stimulator of periodontal ligament cell migration and mitogenesis, but its effect on matrix production is not clear (40) (Table 5).

Platelet-derived growth factor

Ross et al. (711, Kohler & Lipton (51) and Westermark & Wasteson (93) provided evidence for a growth factor derived from platelets, after Balk (2) had pub- lished results showing that whole blood-derived serum was more potent than plasma-derived serum in promoting the growth of chicken fibroblasts at low calcium concentration. Subsequently, the mitogenic activity was called platelet-derived growth factor. Platelet-derived growth factor is a potent growth

Table 4. Effect of selected cytokines on matrix synthesis and remodeling by various cell types. PDGF: platelet-derived growth factor; TGF-aIP: transforming growth factor-a1 p; EGF: epidermal growth factor; FGF: fibroblast growth factor. -: inhibition; 0: no effect; +: slight effect; + +: moderate effect; + + +: strong effect. c: collagenous matrix; nc: non-collagenous matrix

Epithelial cells Fibroblasts Osteoblasts

PDGF ? [-Gzq -1

TGF-P ? -1 - TGF-a ? +

- EGF + + FGF ? - -

~~

Adapted from Lynch & Giannobile (57) with permission.

factor for various connective tissue cells (Table 3) and is released from the a-granules in platelets in conjunction with blood coagulation (48). The platelet-derived growth factor molecule consists of two disulfide-bonded polypeptide chains, denoted A and B. Its molecular weight is 28-35 kDa. Amino acid sequence analysis revealed some homology between the A and B chains, with perfect conservation of the 8 cysteine residues. The heterodimer consisting of a chain A and a chain B is the major platelet-derived growth factor isoform in human platelets (36). The A- and B-chains of dimeric platelet-derived growth factor variants (AA, AB, BB) are encoded by 2 different and independently expressed genes of approxi- mately 20 kbp length, both consisting of 7 analogous exons spaced by differently sized introns. The human A-chain gene has been found on chromosome 7 (51, and the human platelet-derived growth factor B-chain has been located on chromosome 22 (20, 82).

Platelet-derived growth factor binds to specific high-affinity receptors expressed on the surface of various cell types (41), including fibroblasts, glial

Table 5. Effect of selected cytokines on periodontal ligament cells. PDGF: platelet-derived growth factor; TGF-a: transforming growth factor-a; EGF: epidermal growth factor; FGF: fibroblast growth factor. -: inhibition; 0: no effect; +: slight effect; ++: moderate effect; + + +: strong effect

Migration Mitogenesis Matrix synthesis

PDGF F l ++ + + - TGF-a 0

EGF 0 + 0

FGF I +++ I ++ + I0

Adapted from Lynch & Giannobile (57) with permission.

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cells and vascular smooth muscle cells. Two struc- turally related receptors, platelet-derived growth factor R - a and R-P, composed of 1089 and 1106 amino acid residues, respectively, have been identified (16). The molecules belong to the class 111 receptor tyrosine kinase family, consisting of an extracellular lig- and-binding domain, a juxtamembrane stretch, a transmembrane stretch and an intracellular tyrosine kinase effector domain (see Walters in this volume). Both platelet-derived growth factor receptors are synthesized as precursors of lower molecular weight, and subsequently are converted during transport through the cell to cell surface-expressed forms of 170 and 180 kDa for the a and P receptor, respectively. The receptors specifically bind platelet-derived growth factor and no other growth factors. Lig- and-binding leads to increased turnover rate of receptors, and the rapid internalization and degradation of ligand-bound receptors is probably important in regulating the growth stimulus (68).

The effect of platelet-derived growth factor on migration, mitogenesis and matrix production by various cell types is summarized in Tables 2-5. Platelet- derived growth factor is a powerful promoter of cell migration. For optimum stimulation of migration, however, the presence of extracellular matrix is a prerequisite (52). Also, platelet-derived growth factor is a potent mitogen for cells bearing the platelet-derived growth factor receptors. It acts synergistically with other growth factors as a competence factor and nonreciprocally inhibits epidermal growth factor binding to epidermal growth factor receptors (19). Progression of competent cells activated by platelet-derived growth factor from the S-phase through the rest of the cell cycle and into cell division requires the presence of progression growth factors such as insulin or insulin-like growth factor. Platelet-derived growth factor stimulates type V collagen formation in gingival fibroblasts, with a paral- lel drop in collagen type 111 production (93), and collagenase synthesis is enhanced in dermal fibroblasts (4). Platelet-derived growth factor increases cell proliferation in bone cultures, but surprisingly also enhances bone resorption, a process dependent on prostaglandin synthesis (83).

Transforming growth factors

Transforming growth factors are polypeptides isolated from normal and neoplastic tissues, which are known to cause a change in normal cell growth. Originallv, transforming growth factors were iden-

tified by their ability to induce non-neoplastic, surface-adherent, density-dependent, growth-regulated fibroblasts to form anchorage-independent colonies in soft agar cultures. This process appears to be similar to the neoplastic transformation of normal to malignant cells (58). Such transforming growth factors as transforming growth factor-a and -p have been classified according to their relationship to epidermal growth factor (see below).

Transforming growth factor-a is a 50-amino-acid polypeptide that has a molecular weight of 5.6 kDa and shares extensive amino acid homology with epidermal growth factor. It causes similar biological effects, acting through the epidermal growth factor receptor. However, transforming growth factor-a is synthesized primarily by malignant cells. Trans- forming growth factor-p has a molecular weight of 25 kDa and consists of 2 highly conserved identical polypeptides of 112 amino acids. The 2 subunits are linked by disulfide bonds (30). Transforming growth factor-p was originally purified from human placenta, platelets and bovine kidney. Three distinct forms of transforming growth factor-P have been characterized: transforming growth factor-p,, -P, and -P3. A heterodimer, transforming growth factor-

has also been identified. There is extensive amino acid homology among the 3 transforming growth factors. Transforming growth factor-p, and transforming growth factor-0, are 71% homologous with each other and 80% homologous with transforming growth factor-p,. Transforming growth factor+ appears to be synthesized by all normal cells studied to date, and it has been found in many different species. It has been found in higher concentration in the a-granules of platelets, where it is present in amounts equivalent to platelet-derived growth factor. It is secreted in a latent form, and its activities at the target tissue level may reflect the ability of the target tissue to regulate its own growth (72). Three cell surface receptors for transforming growth factor-p have been described, but the mechanisms of signal transduction to effect the expression of target genes are still unclear. The receptors with molecular weights of 53 kDa, 73 kDa and 130 kDa are highly specific and of high affinity. They are down-regulated in the presence of the ligand.

Transforming growth factor+ selectively stimulates the synthesis of connective tissue matrix components, such as collagen (28, 761, fibronectin (86), proteoglycan (14) and glycosaminoglycan (92) both in vitro and in vivo. These effects may be enhanced by reducing the synthesis of proteinases that are in- volved in connective tissue degradation, such as col-


lagenase (701, plasminogen activator or elastase (54). Furthermore, transforming growth factor-p can modify the effects of other growth factors. For example, transforming growth factor-p reduces the level of collagenase expression induced by epidermal growth factor and basic fibroblast growth factor and enhances the induction of tissue inhibitors of metalloproteinases by epidermal growth factor and fibroblast growth factor (26). Transforming growth factor-p has significant effects on bone formation, though its exact role remains elusive. A function in the coupling of bone formation to resorption has been suggested (12). The most important effects of transforming growth factor-a and transforming growth factor-p on various cell types are shown in Tables 2-5.

Interleukin- 1

Interleukin- 1 was initially described as lymphocyte- activation factor, B cell-activating factor, B cell-dif- ferentiating factor or mitogenic protein. In 1978 it was named interleukin- 1 because the term interleukin reflected best its basic property of mediating the communication link among leukocytes. Interleukin- 1 is a polypeptide with a great number of roles in immunity, inflammation, tissue breakdown and tissue homeostasis (63). It is synthesized by various cell types, including macrophages, monocytes, lymphocytes, vascular cells, brain cells, skin cells and fibroblasts, following cellular activation. Induc- tion of interleukin-1 synthesis by such cells can be antigen-dependent or -independent and can also occur following cell stimulation by other cytokines (24). Down-regulation of interleukin- 1 expression has been observed in the presence of cortico- steroids, interleukin-4, interferon-y, prostaglandin E, and histamine.

Unstimulated cells possess low levels of mRNA for interleukin- 1. Gene transcription occurs rapidly after stimulation, and translation into interleukin-1 is observed 15-30 min post-stimulation. Interleukin- 1 is initially synthesized as a 33-kDa precursor molecule that is subsequently processed during or after secretion to smaller entities of 15-17 kDa (33). Two entities of interleukin-1 are best known as interleukin- la and interleukin-lp, two peptides encoded by 2 distinct human cDNAs. Interleukin-la and interleukin-10 share only 27% homology at the amino acid level, but they have similar biological functions. Most human interleukin- 1 produced by stimulated

macrophages is interleukin- lp. Interleukin- la remains largely cell-associated, whereas interleukin- 1 p is released from the cell (39). The two interleukin forms bind to the same receptor, which is found on many cell types in varying densities. Gingival fibroblasts exhibit a higher receptor density than other fibroblasts. The receptor is down-regulated in response to ligand binding (62). Interleukin-1 signal transduction pathways are discussed by Walters in this volume. Regulation of interleukin- 1 synthesis is secured through several pathways, of which synthesis of prostaglandin E, appears to be of great importance (53). In addition, several natural inhibitors of interleukin- 1 activities have been identified and described (56).

Interleukin-1 mediates tissue remodeling, repair and inflammation through many physiological and pathological processes. Unrestricted production of interleukin-1 may lead to severe tissue damage. Thus, interleukin-1 was suggested to play a key role in the pathogenesis of bone diseases and adult periodontitis (84). The numerous biological activities of interleukin- 1 have been reviewed extensively (25).

With regard to homeostasis of periodontal tissue, stimulation of the proliferation of keratinocytes, fibroblasts and endothelial cells is important. Furthermore, interleukin- 1 enhances fibroblast synthesis of type I procollagen, collagenase, hyalu- ronic acid, fibronectin and prostaglandin E,. Also, it has been found that interleukin-1 stimulates bone resorption and is identical to osteoclast-activating factors (33).

Interferon-y

Interferon was discovered in 1957 by Isaacs & Lin- denmann (44). They found a protein that could con- fer on cells resistance to a variety of viruses. Sub- sequent further analysis revealed 3 functionally related groups of protein, designated interferon-a, -J3 and -y (78). Interferon-y can be considered as a distinct entity compared with the other interferons. It possesses important immunomodulatory effects and thus is a lymphokine as much as an interferon. This review is limited to the description of interferon-y.

The original determination of the molecular weight of human interferon-y obtained from mitogen-stimulated peripheral blood leukocytes suggested 35-70 kDa. Further analysis, however, revealed 2

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Table 6. Induction and repression of matrix metalloproteinases. Adapted from Birkedal-Hansen (7) with permission

Metalloproteinase expression Inducers Rewessors

~~

Interleukin-l (29, 58) Tumor necrosis factor-a (8, 22, 58) TGF-a and -p (6, 73) Epidermal growth factor (50) Platelet-derived growth factor (4, 50) Basic fibroblast growth factor (3 , 26) Prostaglandin E, (88) Parathyroid hormone (15)

Interferon-y ( 1 , 89) Transforming growth factor-P (26, 50) Glucocorticoids (29, 47) Progesterone (67)

proteins of 20 and 25 kDa. Interferon-y has very little structural homology with interferon-a and interferon-b. The interferon-y gene has 4 exons and is located on chromosome 12 in humans. The gene codes for 166 amino acids, but the first 23 amino acids act as a signal sequence and are cleaved from the molecule during secretion. The secreted interferon-y has 143 amino acids and exhibits two glycos- ylation sites, which are located at residues 25 and 97. The presence of carbohydrate groups gives raise to the two natural forms of 20 kDa and 25 kDa, which differ biochemically but not in their functional properties (95). Interferon-y synthesis in and secretion by TH-1 lymphocytes can be induced by such substances as mitogens, antigens or antibodies directed against lymphocyte surface antigens. The production of interferon-y is modulated by other cytokines such as interleukin-1.

Specific receptors for interferon-y have been characterized on a great variety of cell types. The molecular weight of receptors on monocytes and hematopoi- etic cell lines is 140 kDa, whereas it is 95 kDa on other cells, such as fibroblasts. The signal produced by ligand binding to the receptor leads to rapid internalization and receptor down-regulation, but other stimuli seem to be necessary to induce cell activation (55). Following receptor binding, interferon-y initiates reactions leading to gene expression.

Many biological activities have been ascribed to interferon-y, including action on B and T lymphocytes, antibody production, natural killer cells, macrophages and tumor cells. Antiviral activities have been observed for interferon-y, but these are less pronounced as compared with interferon-a and interferon-p (23). Besides such cellular reactions, interferon-y affects collagen production by turning off collagen gene expression.

Matrix metalloproteinases and their tissue inhibitors

There is strong evidence from in vitro and in vivo studies that connective tissue cells participate in both the formation and the breakdown of connective tissue matrix (65). Such cells were found to syn- thesize and secrete a family of enzymes collectively known as matrix metalloproteinases. The proteinases share in common that they function at neutral pH, can probably digest all the major matrix macro- molecules and contain a Zn2+ binding site within the catalytic domain of the molecule. The role of matrix metalloproteinases in health and disease has been the subject of extensive investigation and has been reviewed in detail recently (6, 7).

The various matrix metalloproteinases share extensive amino acid homology with regard to their 5- domain chemical structure. However, they differ substantially in their molecular weight because of the addition or deletion of domains. For example, collagenase obtained from polymorphonuclear leukocytes (matrix metalloproteinase-8) is consider- ably larger (molecular weight 75 kDa) than collagenase secreted by fibroblasts (matrix metalloproteinase-1; molecular weights 52 and 57 kDa), but they degrade similar substrates. Furthermore, as outlined by Birkedal-Hansen (6), the two interstitial collagen- ases differ significantly with regard to gene transcription. Neutrophil collagenase is released almost instantaneously upon challenge, whereas it takes fibroblasts several hours to release the enzyme. Also, transcription of fibroblast matrix metalloproteinase genes is subtly regulated by a variety of cytokines and growth factors (Table 61, but a comparable regulation is not known for neutrophil collagenase. Mat- rix metalloproteinases are synthesized as latent pre-

70

Aspects of cell biology of the normal periodontium

cursor molecules. The latency is likely due to the folding of the cysteine residue at position 73 of the proenzyme over the Zn2+ binding site. The binding of Cys-Zn2+ effects a conformational change in the molecule's structure, and enables the binding of an H,O molecule to the Zn2+ site (78). The fully active enzyme is obtained following cleavage of the Cys- Zn2+ bond and autolytic or other enzymatic modifi- cations (35, 81). Although activation steps can be reasonably explained in vitro, the mechanisms in- volved in uivo are insufficiently understood (6).

The observations reported on the effects of transforming growth factor-p on matrix metalloproteinase regulation are significant with regard to tissue homeostasis. This cytokine is of special interest because, in contrast to most cytokines, it can repress matrix metalloproteinase production. However, it has been shown that expression of the 92-kDa keratinocyte gelatinase (matrix metalloproteinase-9) is turned on by transforming growth factor+, whereas fibroblast gelatinase (matrix metalloproteinase-2) production is turned off by transforming growth factor$ (70, 73) . Metalloproteinases have been de- tected in gingival crevicular fluid (871, saliva (21) and plasma (96).

The activity of matrix metalloproteinases is regulated further by serum-derived a-macroglobulin and by tissue inhibitors of metalloproteinases. a-Macro- globulin is a very large molecule with a molecular weight of 725 kDa, and in its native state, it consists of four 185-kDa polypeptide subunits. In addition to matrix metalloproteinases, a-macroglobulin inter- acts with a large number of cytokines and may inter- fere with their release, activation and degradation (45). It is found in high concentrations in gingival crevicular fluid (74). Tissue inhibitors of metalloproteinases represent a family of at least 2 polypeptides, tissue inhibitor of metalloproteinase-1 and -2. Tissue inhibitor of metalloproteinase-1 has a molecular weight of 28 kDa and forms complexes with active matrix metalloproteinase-1 but not with its precursor (91). Tissue inhibitor of metalloproteinase-2 is a slightly smaller molecule (22 kDa) that inhibits matrix metalloproteinase-2 via complex formation (34). Tissue inhibitors of metalloproteinases are expressed by various connective tissue cells (801, epithelial cells (6), endothelial cells (10) and macrophages (43). Cytokines selectively affect the expression of the tissue inhibitors of metalloproteinase-1 and -2 genes, which are located on the X chromosome and chromosome 17, respectively. Tissue inhibitor of metalloproteinase- 1 gene expression is enhanced in the presence of epidermal

growth factor, tumor necrosis factor-a, interleukin- 1 and transforming growth factor$. In contrast, transforming growth factor-p represses the gene expression of tissue inhibitor of metalloproteinase-2 (6) .

The role of cytokines, matrix metalloproteinases and tissue inhibitors of metalloproteinases in connective tissue homeostasis

Connective tissue homeostasis involves a multitude of perfectly coordinated anabolic and catabolic processes at the cellular and extracellular level (37). In fact, cellular homeostasis in tissues is likely to be the result of a balance among complex interactions of antagonistic and synergistic molecules, with cytokines, matrix metalloproteinases and natural inhibitors of matrix metalloproteinase playing major roles. The literature on cytokines is growing rapidly, and despite the great wealth of available information, the understanding of the role of individual compounds is still vague and deficient. Clearly, it is not possible to single out one most important pathway of tissue homeostasis. A major problem in cytokine biology is redundancy. Some cells seem to be influenced by many cytokines, but others demand specific influ-

Fig. 1. Schematic model of connective tissue homeostasis, exhibiting cells in proliferation, aptotic cells, cells produc- ing matrix proteins and cells degrading their extracellular matrix. Such cell activities are modulated by various cytokines and other soluble cellular products. PDGF: platelet- derived growth factor. TGF-J3: transforming growth factor- p. FGF: fibroblast growth factor. INF-y: interferon-y. TNF: tumor necrosis factor. IL-1: interleukin- 1. PGE,: prostaglandin E,. EGF: epidermal growth factor. TIMP: tissue inhibitors of metalloproteinases. MMP: matrix metalloproteinases.

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Hefii

ence by one cytokine only. Likewise, some cytokines have a broad spectrum of target cells, whereas others act on one cell type only. In addition, individual cell responses to cytokine stimulation vary greatly (Tables 2-5). Yet another factor of importance is that, in contrast to most studies i12 vim, cytokines do not occur as single entities iii zlivo. Only recently, results have become available from studies that combined several cytokines to affect cellular targets (40, 57). Considering all these and similar deficiencies, I would like to propose a possible scenario of the cytokine-mediated regulation of periodontal tissue homeostasis, as depicted in Fig. 1.

The model suggests the simultaneous existence of 4 different functional stages and phenotypes (70) of fibroblast cells: 1) a cell in proliferation; 2) a cell that participates in matrix synthesis and secretion; 3) a cell that participates in matrix resorption; and 4) a cell in apoptosis. Each phenotype is characterized by the expression of specific genes induced by a cytokine or combinations of several cytokines. Platelet- derived growth factor, fibroblast growth factor and interleukin- 1 have been characterized as being inducers of anabolic cell activities, whereas interferon- y and prostaglandin E, are inhibitors of such activities. Of great interest in this context is transforming growth factor+, which has been shown (70) to increase the synthesis of functional tissue inhibitors of metalloproteinases in gingival fibroblasts while reducing collagenase expression by the same cells. Such reciprocity assigns transforming growth factor-

an exquisite role in extracellular matrix homeostasis. Apoptotic death of connective tissue cells is poorly understood and needs further investigation. It has been suggested that cytokines may constitute the external signal that triggers the “suicide pathway” (18).

Acknowledgements

Arthur F. Hefti received support from USPHS grant DE-07481 from NIDR during preparation of this art- icle. The author thanks Ms. Janice R. Braddy for manuscript preparation.

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