Periodontology 2000, Val. 3.
1993, 64-75
Printed in Denmark . All righrs resented
Aspects of cell biology of the
normal periodontium
ARTHURF. 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 peri-
odontal 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 peri-
odontal tissues are among the most biologically ac-
tive in the body, exhibiting a remarkably high turn-
over 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 an-
tagonistic 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 peri-
odontal diseases (31). Growth factors and cytokines
that are believed to play primary roles in the patho-
genesis 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 for-
mation 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-
64
Copyright 0 Munksgaard 1993
PERIODONTOLOGY 2000
ISSN 0906-6713
nective tissue remodeling and turnover? There is
abundant evidence that cytokines, which are se-
creted by fibroblasts (641, endothelial cells (751, epi-
thelial cells and cells of the immune system (91, play
a decisive role in tissue homeostasis, acting in con-
cert with matrix metalloproteinases and their natu-
ral inhibitors, tissue inhibitors of metalloproteinas-
es. 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 ob-
served 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 fac-
tors 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 de-
scribed (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 var-
iety 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 de-
rived from monocytes, which caused lymphocyte ac-
tivation, appeared to be chemically identical to the
activity that previously was known as B-cell activat-
ing factor or mononuclear cell factor. In 1978, the
 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 in-
terleukin-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 poly-
peptides and glycoproteins that are secreted by one
or several cell types and act locally or systemically.
Included in the cytokine molecule group are in-
terleukins, cytotoxic factors, interferons, growth fac-
tors, colony-stimulating factors and intercrines (42).
A comprehensive list of human cytokines, their mol-
ecular 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 17.5 159 monocyte/macrophage,
endothelial cell, fibroblast
Interleukin-l p 17.5 153 keratinocyte
Interleukin-2 15-20 133 T-cell
Interleukin-3 14-30 133 T-cell, mast cell
Interleukin-4 15-19 129 T-cell, mast cell
Interleukin-5 2X21.5 2x113 T-cell, mast cell
Interleukin-6 21-28 184 macrophage, fibroblast, T-cell,
endothelial cell
Interleukin-7 25 152 bone marrow cells
Interleukin-8 6-10 72 monocyte, fibroblast, endothelial cell,
keratinocyte
Interleukin-9 32-39 179 T-cell
Interleukin-10 (murine) 19 160 T-cell
Interleukin-11 23 199 bone marrow cell
Interleukin-12 70 B-cell
Cytotoxic factors
Tumor necrosis factor-a 17 157 monocyte/macrophage, fibroblast,
T-cell
Tumor necrosis factor-p 25 171 T-cell, B-cell
Interferons
Interferon-a 16-27 166 macrophage
Interferon$ 20 166 fibroblast
Interferon-y 20-25 146 T-cell, natural killer cell
Growth factors
Epidermal growth factor 6 53 macrophage
Acidic fibroblast growth factor 15-18 149 platelet, macrophage
Basic fibroblast growth factor 15-18 146 endothelial cell
Insulin-like growth factor-1 7 70 macrophage
Platelet-derived growth factor-1 28-35 2x104 platelet, monocyte/macrophage,
endothelial cell
Transforming growth factor-a 5-6 50 macrophage
Transforming growth factor-p 25 2x112 platelet, monocyte/macrophage, T-cell,
fibroblast, endothelial cell
Colony-stimulating factors
Granulocyte colony-stimulatingfactor 20 177 monocytelmacrophage, fibroblast,
endothelial cell
Granulocyte-macrophage 22 127 T-cell, fibroblast, endothelial cell
colony-stimulating factor
Macrophage colonykmulating factor 70-90 224 fibroblast. monocvte. endothelial cell
, I

65
Hefti
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 pres-
ence of other cytolunes, the state of cell activation
and the degree of cell differentiation. In vivo, cyto-
kines are believed to play an important role in nu-
merous 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 compre-
hensive 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 fibro-
blast growth factor was discovered by its ability to
induce proliferation in mouse fibroblasts; acidic
fibroblast growth factor was found to delay differen-
tiation of myoblasts and to stimulate endothelial cell
proliferation. The two fibroblast growth factors are
products of different genes (46, 60, 75) but are simi-
lar in structure and biological activities. Both fibro-
blast 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, respec-
tively. Their expression appears to be independently
regulated. The fibroblast growth factor mRNA levels
in cells are normally very low, possibly because of
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. -: inhi-
bition; 0: no effect; +: slight effect; + +: moderate effect; + + +: strong effect
Epithelial cells Fibroblasts
PDGF 0 -+++I
TGF-cx F l + +
TGF-P + +
EGF + 0
FGF F
l 1-
Adapted from Lynch & Giannobile (57) with permission.
66
mRNA instability, but tissue levels are relatively
high. The predominant view is that fibroblast growth
factors are not humoral factors but rather locally ac-
tive 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 fac-
tors suggest an interaction with their surface recep-
tors 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 pro-
tein synthesis ( l l ) ,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
Endothelial cells Inflammatory cells Osteoblasts
0 01+ 1(
0 ?
? /TTl ?
0 ?
1- 0 ?
 Aspects of cell biology of the normal periodontiurn

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. -: inhi-
bition; 0: no effect: +: slight effect; ++: moderate effect; +++: strong effect ~~ ~~ ~

Epithelial cells Fibroblasts Endothelial cells Smooth muscle Osteoblasts

cells
PDGF 0 pq 0 p 7 - l F]
TGF-a FJ + + ? +
TGF-D - +I- - ? +
EGF I +++ I + + ? +
FGF pq ++ Vl + +
Adapted from Lynch & Giannobile (57) with permission.

between the actions of acidic fibroblast growth fac-
tor and basic fibroblast growth factor is that the ef-
fect of basic fibroblast growth factor on cell repli-
cation 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 mitogen-
esis, 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 fac-
tor 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 mito-
genic activity was called platelet-derived growth fac-
tor. Platelet-derived growth factor is a potent growth
factor for various connective tissue cells (Table 3 )
and is released from the a-granules in platelets in
conjunction with blood coagulation (48). The plate-
let-derived growth factor molecule consists of two
disulfide-bonded polypeptide chains, denoted A and
B. Its molecular weight is 28-35 kDa. Amino acid se-
quence 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 differ-
ent and independently expressed genes of approxi-
mately 20 kbp length, both consisting of 7 analogous
exons spaced by differently sized introns. The hu-
man 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 4. Effect of selected cytokines on matrix syn-

thesis and remodeling by various cell types. PDGF: Table 5. Effect of selected cytokines on periodontal
platelet-derived growth factor; TGF-aIP: transforming ligament cells. PDGF: platelet-derived growth factor;
growth factor-a1p; EGF: epidermal growth factor; FGF: TGF-a: transforming growth factor-a; EGF: epidermal
fibroblast growth factor. -: inhibition; 0: no effect; +: growth factor; FGF: fibroblast growth factor. -: inhi-
slight effect; + +: moderate effect; + + +: strong effect. bition; 0: no effect; +: slight effect; ++: moderate ef-
c: collagenous matrix; nc: non-collagenous matrix fect; + + +: strong effect
Epithelial cells Fibroblasts Osteoblasts Migration Mitogenesis Matrix
synthesis
PDGF ? [-Gzq1 -
TGF-a ? + - PDGF F l ++ +
TGF-P ? 1- TGF-a 0 - +
EGF + + - EGF 0 + 0

FGF ? - - FGF I +++ I ++ + I0

~~

Adapted from Lynch & Giannobile (57) with per- Adapted from Lynch & Giannobile (57) with per-
mission. mission.

67
Hefti
cells and vascular smooth muscle cells. Two struc-
turally related receptors, platelet-derived growth fac-
tor 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 tyro-
sine 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, respec-
tively. The receptors specifically bind platelet-de-
rived growth factor and no other growth factors. Lig-
and-binding leads to increased turnover rate of
receptors, and the rapid internalization and degra-
dation of ligand-bound receptors is probably im-
portant in regulating the growth stimulus (68).
The effect of platelet-derived growth factor on mi-
gration, mitogenesis and matrix production by vari-
ous 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-de-
rived growth factor receptors. It acts synergistically
with other growth factors as a competence factor
and nonreciprocally inhibits epidermal growth fac-
tor 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 divi-
sion requires the presence of progression growth
factors such as insulin or insulin-like growth factor.
Platelet-derived growth factor stimulates type V col-
lagen formation in gingival fibroblasts, with a paral-
lel drop in collagen type 111 production (93),and col-
lagenase synthesis is enhanced in dermal fibroblasts
(4). Platelet-derived growth factor increases cell pro-
liferation in bone cultures, but surprisingly also en-
hances bone resorption, a process dependent on
prostaglandin synthesis (83).
Transforming growth factors
Transforming growth factors are polypeptides iso-
lated 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, sur-
face-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 fac-
tors as transforming growth factor-a and -p have
been classified according to their relationship to epi-
dermal 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 epi-
dermal growth factor. It causes similar biological ef-
fects, acting through the epidermal growth factor re-
ceptor. 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 pla-
centa, 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 trans-
forming growth factor-p,. Transforming growth fac-
tor+ appears to be synthesized by all normal cells
studied to date, and it has been found in many dif-
ferent species. It has been found in higher concen-
tration 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 mech-
anisms of signal transduction to effect the ex-
pression of target genes are still unclear. The recep-
tors 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 stimu-
lates the synthesis of connective tissue matrix com-
ponents, 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 ex-
ample, transforming growth factor-p reduces the
level of collagenase expression induced by epider-
mal 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 interleu-
kin 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 ob-
served 15-30 min post-stimulation. Interleukin- 1 is
initially synthesized as a 33-kDa precursor molecule
that is subsequently processed during or after secre-
tion to smaller entities of 15-17 kDa (33). Two enti-
ties of interleukin-1 are best known as interleukin-
la and interleukin-lp, two peptides encoded by 2
distinct human cDNAs. Interleukin-la and in-
terleukin-10 share only 27% homology at the amino
acid level, but they have similar biological functions.
Most human interleukin- 1 produced by stimulated
Aspects of cell biology of the normal periodontiurn
macrophages is interleukin- lp. Interleukin- la re-
mains largely cell-associated, whereas interleukin- 1p
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 fibro-
blasts exhibit a higher receptor density than other
fibroblasts. The receptor is down-regulated in re-
sponse 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 syn-
thesis of prostaglandin E, appears to be of great im-
portance (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 ex-
tensively (25).
With regard to homeostasis of periodontal
tissue, stimulation of the proliferation of keratino-
cytes, 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-activ-
ating 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 inter-
feron-y.
The original determination of the molecular
weight of human interferon-y obtained from mitog-
en-stimulated peripheral blood leukocytes suggested
35-70 kDa. Further analysis, however, revealed 2
69
Hefii
Table 6. Induction and repression of matrix metalloproteinases. Adapted from Birkedal-Hansen (7) with per-
mission
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)
proteins of 20 and 25 kDa. Interferon-y has very little
structural homology with interferon-a and inter-
feron-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 inter-
feron-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 func-
tional 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 char-
acterized on a great variety of cell types. The molecu-
lar 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 li-
gand binding to the receptor leads to rapid internaliz-
ation and receptor down-regulation,but other stimuli
seem to be necessary to induce cell activation (55).
Following receptor binding, interferon-y initiates re-
actions leading to gene expression.
Many biological activities have been ascribed to
interferon-y, including action on B and T lympho-
cytes, 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, in-
terferon-y affects collagen production by turning off
collagen gene expression.
70
Interferon-y ( 1 , 89)
Transforming growth factor-P (26, 50)
Glucocorticoids (29, 47)
Progesterone (67)
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 connec-
tive tissue matrix (65). Such cells were found to syn-
thesize and secrete a family of enzymes collectively
known as matrix metalloproteinases. The protein-
ases 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 ex-
tensive 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 collagen-
ase secreted by fibroblasts (matrix metalloprotein-
ase-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 tran-
scription. 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 regu-
lation is not known for neutrophil collagenase. Mat-
rix metalloproteinases are synthesized as latent pre-

cursor molecules. The latency is likely due to the
folding of the cysteine residue at position 7 3 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 trans-
forming growth factor-p on matrix metalloproteinase
regulation are significant with regard to tissue
homeostasis. This cytokine is of special interest be-
cause, in contrast to most cytokines, it can repress
matrix metalloproteinase production. However, it
has been shown that expression of the 92-kDa kera-
tinocyte gelatinase (matrix metalloproteinase-9) is
turned on by transforming growth factor+, whereas
fibroblast gelatinase (matrix metalloproteinase-2)
production is turned off by transforming growth fac-
tor$ (70, 7 3 ) . Metalloproteinases have been de-
tected in gingival crevicular fluid (871, saliva (21) and
plasma (96).
The activity of matrix metalloproteinases is regu-
lated 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 metallo-
proteinases represent a family of at least 2 polypep-
tides, tissue inhibitor of metalloproteinase-1 and -2.
Tissue inhibitor of metalloproteinase-1 has a mol-
ecular weight of 28 kDa and forms complexes with
active matrix metalloproteinase-1 but not with its
precursor (91). Tissue inhibitor of metalloprotein-
ase-2 is a slightly smaller molecule (22 kDa) that in-
hibits matrix metalloproteinase-2 via complex for-
mation (34). Tissue inhibitors of metalloproteinases
are expressed by various connective tissue cells (801,
epithelial cells (6), endothelial cells (10) and macro-
phages (43). Cytokines selectively affect the ex-
pression of the tissue inhibitors of metalloprotein-
ase-1 and -2 genes, which are located on the X
chromosome and chromosome 17, respectively.
Tissue inhibitor of metalloproteinase- 1 gene ex-
pression is enhanced in the presence of epidermal
Aspects of cell biology of the normal periodontium
growth factor, tumor necrosis factor-a, interleukin- 1
and transforming growth factor$. In contrast, trans-
forming growth factor-p represses the gene ex-
pression 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 pro-
cesses 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 cyto-
kines, matrix metalloproteinases and natural inhibi-
tors 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 cyto-
kines 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,: prosta-
glandin E,. EGF: epidermal growth factor. TIMP: tissue in-
hibitors of metalloproteinases.MMP: matrix metallopro-
teinases.
71
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 cyto-
kine or combinations of several cytokines. Platelet-
derived growth factor, fibroblast growth factor and
interleukin- 1 have been characterized as being in-
ducers of anabolic cell activities, whereas interferon-
y and prostaglandin E, are inhibitors of such activi-
ties. Of great interest in this context is transforming
growth factor+, which has been shown (70) to in-
crease the synthesis of functional tissue inhibitors of
metalloproteinases in gingival fibroblasts while re-
ducing collagenase expression by the same cells.
Such reciprocity assigns transforming growth factor-
an exquisite role in extracellular matrix homeo-
stasis. 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 path-
way” (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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