Archs oral Bid. Vol. 36, No. 11, pp. 191-191, 1991 F’rintcd in Great Britain. All rights reserved
ooo3-9969/91 S3.00 + 0.00 Copyright 0 1991 Pcrgamon Press plc
LIPOPOLYSACCHARIDE STIMULATION OF HYALURONATE SYNTHESIS BY HUMAN GINGIVAL FIBROBLASTS IN VITRO P. M. BARTOLD Department of Pathology, University of Adelaide, Adelaide, Australia (Accepted 31 May 1991) Summary-Exposure of gingival fibroblasts to LPS caused a dose-dependent increase in hyaluronate synthesis. Stimulation of hyaluronate synthesis by LPS was significantly greater 24 h after exposure and by 48 h an approx. 50% increase was evident. In parallel, there was an increase in the activity of the hyaluronate synthetase enzyme. Inhibition of PGE, synthesis by indomethacin abolished the stimulatory effect of LPS on hyaluronate synthesis. Thus, this stimulatory effect of LPS on hyaluronic acid synthesis may be a secondary response to the induction of PGE,. The molecular size of newly synthesized hyaluronate was not affected by LPS. The metabolic changes observed may be a primary response of the cells to bacterial toxins and may aid extracellular matrix repair. Key words: hpopolysaccharide,
hyaluronate, fibroblasts.
INTRODUCTION Lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria, has received considerable attention in relation to the pathogenesis of the periodontal diseases (Daly, Seymour and Kieser, 1980). In particular, it has been shown to
modulate cell growth (Vaheri et al., 1973; Larjava et al., 1987), stimulate bone resorption (Hausman, Raisz and Miller, 1970; Millar et al., 1986) and induce lymphokine release by macrophages (Hanazawa et al., 1985). Thus, it is considered to be a potent biological agent capable of important effects in infected tissues either by direct influences on cell metabolism, or, indirectly, by invoking an inflammatory response. The connective tissues of the periodontium have both fibrous and non-fibrous elements which, together, impart structural and physiological properties that allow adequate function (Narayanan and Page, 1983; Bartold, 1987). However, upon induction of inflammation, numerous changes occur within the extracellular matric.es of the periodontium (Page and Schroeder, 1976). In addition to ultrastructural changes, the metabolism of the resident fibroblasts is also disturbed. The combination of tissue destruction and altered cellular activity leads to compromised function. In recent years, the metabolism and degradation of periodontal collagens has been well studied (Narayanan and Page, 1983). However, other principal components of the extracellular matrix such as proteoglycans and hyaluronate have received less attention (Bartold, 1987). Therefore, as hyaluronate has been implicated in critical functions such as the Abbreviarions: CPC’, cetylpyridinium chloride; DVM, Dulbecco-Vogt medium; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; PG, prostaglandin.
regulation of cell-cell interactions, tissue hydration, and control of macrophages and lymphocytes (Comper and Laurent, 1978), my purpose now was to investigate the effect of LPS on hyaluronate synthesis by human gingival fibroblasts in vitro. MATERIALS
AND METHODS
Materials
Guanidine HCl, indomethacin, glutamine, adenosine 5’ triphosphate (ATP), and UDP-N-acetylglucosamine were all purchased from the Sigma Chemical Co., St Louis, MO, U.S.A. CPC was from Ajax Chemicals Australia, Aulam, N.S.W.; filter paper (3MM) from Whatman, Maidstone, U.K.; Streptomyces hyaluronidase from Seikagaku Kogyo Ltd, Tokyo, Japan; Streptomyces griseus pronase from Calbiochem, San Diego, CA; Sephadex G-50 and Sepharose CL-2B from Pharmacia Fine Chemicals, North Ryde, Sydney, Australia; DVM, fetal bovine serum, penicillin, streptomycin and nonessential amino acids were all from Flow Laboratories, Irvine, Ayrshire, U.K.; tissue-culture ware was from Nunc, Roskilde, Denmark; o-[6-3H]-g1ucosamine HCl(33 Ci/mmol) and UDP-D-[U-14C]-glucuronic acid (225 mCi/mmol) were from Amersham (Australia), Sydney, Australia; Ready Solv EP scintillation fluid was from Beckman (Australia), Adelaide, Australia. LPS preparation
LPS from Actinobacillus actinomycetemcomitans was a gift from Dr E. Hausman’s laboratory, State University of New York at Buffalo and had been extracted by the phenol-water method (Westphal and Jann, 1965). Although no subsequent purification was carried out, and it was considered a crude LPS preparation, it has significant biological activity 791
792
P.M. BARTOLD
attributable to LPS (Bartold and Millar, 1988). LPS from Salmonella enteritidis was purchased from Sigma Chemical Co., St Louis, MO, U.S.A. It had been extracted with phenol and purified by gel filtration chromatography. Both its protein and RNA contents were less than 1%. Fibroblast cultures Fibroblasts were obtained by explant culture from biopsies of healthy human gingiva from volunteers, as described by Narayanan, Page and Kuzan (1978); these have been used extensively in previous studies (Bartold and Page, 1986a; Bartold and Millar, 1988). The cells were maintained in DVM supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml of penicillin, 100 pg/ml streptomycin, 2 mM glutamine and non-essential amino acids at 37°C in an atmosphere of 5% CO, and 95% air. Cells between the 4th and 8th transfer in culture were used. All experiments, unless otherwise stated, were carried out in complete DVM containing 10% fetal bovine serum. Radioisotope incorporation studies Initial studies were designed to investigate the effect of various preparations of LPS on hyaluronate synthesis by the fibroblasts. Fibroblasts were seeded in triplicate at a concentration of 5 x lo4 cells per well, into 24-well plates. Upon reaching confluence (1 x lo5 cells/well), the cells were incubated in medium containing LPS from either S. enteritidis or A. actinomycetemcomitans ranging from 50 to 0.5 pg/ml for 24 h and pulsed with [3H]-glucosamine (20 pCi/ml) for the entire period. The medium was then removed and the cell layers washed once with PBS, which was pooled with the medium to make up a fraction for subsequent analyses of hyaluronate content. The cells were released from the culture plates by trypsinization. A sample was taken for counting cell numbers in a haemocytometer and the remainder was frozen until required. To determine the effect of LPS on the rate of hyaluronate synthesis, confluent cells were cultured as described above. Upon reaching confluence the medium was removed and the cells incubated in the presence or absence of 50pg/ml S. enteritidis LPS for 4, 8, 24, 30 48 and 12 h. The incorporation of [‘HI-glucosamine into hyaluronate was determined by selective digestion with hyaluronidase for each time point after the introduction of the radiolabel and 50 pg/ml LPS. As PGE? can influence hyaluronate synthesis and because LPS has been reported to stimulate prostaglandin synthesis in other systems (Yaron et al., 1978; Wahl et al., 1977) the effect of S. enteritidis LPS on hyaluronate synthesis in the absence of endogenous PGE, was investigated. Gingival fibroblasts were grown to confluence in 24-well plates and incubated in the presence or absence of 50 pg/ml LPS in DVM containing 20 pCi/ml [3H]-glucosamine for 24 h. To inhibit PGE, synthesis, indomethacin ranging in concentration from 10m6 to 10m4M was also added to some cultures. This concentration range inhibited PGE, synthesis by fibroblasts in culture (Dayer et al., 1979) and was without toxic effects on human
gingival fibroblasts (Bartold, 1988). Hyaluronate synthesis was monitored by hyaluronidase digestion of molecules labelled with [3H]-glucosamine. To obtain material for molecular-size analysis, human gingival fibroblasts were grown to confluence in 25-cm* culture flasks. Upon attaining confluence (5 x lo5 cells per flask) the culture medium was removed and the cells cultured in the presence or absence of 5Opg/ml S. enteritidis LPS for 24 h in DVM containing 20 pC!i/ml [‘HI-glucosamine. After incubation the medium was removed and the cell layers washed with 3 ml PBS which was then pooled with the medium to make up this fraction. The remaining cells in culture vessels were then extracted with 5 ml of 4 M guanidine HCl/O.OS M sodium acetate, pH 5.8, overnight at 4°C. After 24 h at 4°C this cell fraction was centrifuged to remove any debris and the supematant used for subsequent analyses as the cell-layer fraction. Both the medium and cell-layer extracts were desalted by gel filtration chromatography on Sephadex G-50 to remove unincorporated [3H]-glucosamine (Bartold and Page, 1986a). The labelled macromolecular material which eluted in the void volume of Sephadex G-50 (1 x 30 cm) was used for subsequent analyses of hyaluronate molecular size. Determination of 3H-labelled hyaluronate Hyaluronate synthesis by human gingival fibroblasts in vitro was determined as described previously (Castor et al., 1981; Huey, Stair and Stem, 1990). In brief, samples (200 ~1) from the culture medium were digested with pronase (10 mg/ml) overnight at 60°C. They were then boiled and split into two portions, each of 50 ~1. One portion (designated B) was incubated at 37°C for 12 h with 60~1 Streptomyces hyaluronidase in 0.1 M sodium acetate buffer at pH 6.0. The other (designated A) was incubated in the sodium acetate buffer alone under identical conditions. After the incubation, samples (100 ~1) from each part were spotted onto filter paper divided into 2-cm squares, allowed to dry at 60°C and then placed through four washes, each of 30-min duration, in 0.05 M sodium chloride containing 0.1% CPC at room temperature (22°C). The filter paper was then removed and allowed to dry. Each square was then cut into small strips and placed in 14ml of scintillation fluid before determining the radioactivity in a liquid scintillation counter. The amount of hyaluronate in each sample was calculated as the amount of radioactivity digested by the Streptomyces hyaluronidase (i.e. dis/min in portion A - dis/min in portion B. Pulse-chase experiments To determine whether or not the effect of LPS on hyaluronate synthesis was due to an increase in rate of synthesis or decrease in rate of breakdown, pulsechase experiments were made. Fibroblasts were seeded, in triplicate at an initial density of 5 x lo4 cells per well, and allowed to grow to confluency (1 x 10’ cells/well). The confluent monolayers were then incubated in the presence of 2Opg/ml [‘HIglucosamine and 50/rg/ml LPS for 24 h, washed four times with PBS and then chased in 500~1 of
793
LPS and fibroblast hyaluronate
isotope-free medium containing 50 pg/ml LPS for various times. At each time, the medium was removed and the cell layers scraped from the plates. Hyaluronic determinations were carried out on both the medium and &-layer fractions. Hyah4ronate synthetase activity
Molecular-sieve chromatography
The molecular size of hyaluronate synthesized in the presence or absence of S. enteritidis LPS was assessed after 48 h in culture using Sepharose CL-ZB, as described by Bartold and Page (1986a). Approximately the same am’ounts (40,000 dis/min) of radiolabelled macromolecular material isolated from the medium and cell-layer fractions by Sephadex G-50 chromatography were eluted from Sepharose CL-2B (0.7 x 100 cm) with 4 M guanidine HCl in 0.05 M sodium acetate, pH 5.8. With a flow rate of 3 ml/h, fractions of 0.5 ml were collected and 50-~1 samples were taken for radioactivity determination in a liquid scintillation counter. The material which eluted in the void volume was pooled separately from that which eluted in the included volume. These two pools were indicative of the two major molecular-size fractions of [3H]-glucosamine-labelled material eluting from Sepharose CL-2B. After dialysis against 0.1 M
c I m
-
0
Statistical analysis
All data were subjected to analysis of variance.
Gingival fibroblasts were grown to confluence in 24-well plates and cultured in the presence or absence of 50 pg/ml S. enteritidis LPS for 8,24 and 48 h. The cells were removed from the culture surface by trypsinization and the cell pellet obtained by gentle centrifugation was washed twice with PBS before freezing at -70°C. The cells were lysed by freezing and thawing the pellet three times. Hyaluronate synthetase activity was determined by incubating the lysed cells in the presence of 0.1 PCi UDP-D-[U-‘~C]glucuronic acid and 5 x 10-3 PM UDP-N-acetylglucosamine as substrates (Appel, Horwitz and Dorfman, 1979). The: reaction mixture was incubated for 90 min at 37°C and the amount of hyaluronate thus synthesized was determined by selective digestion with hyaluronidase as described above.
3000 = .I-_E .$2000 =In 8 m0 1000
sodium acetate, the hyaluronate content in these fractions was determined by selective digestion with Streptomyces hyaluronidase (Bartold and Page, 1986a).
m Control q se. n A.&
RESULTS The initial experiments to determine the biological effect of LPS on hyaluronic acid synthesis (Fig. l), showed that, in all cases, exposure of the fibroblasts to higher (5 and SOpg/ml) concentrations of LPS increased that synthesis. Concentrations of LPS up to 50 pg/ml had no visible effect on the cells whereas at concentrations of 500 pg/ml, detachment and death occurred (data not shown). Cell counts after the experiments showed no significant change between LPS-treated and -untreated cells (data not shown). Regardless of the source of the LPS, the greatest stimulation of hyaluronate production occurred at the highest concentration which did not cause cell death (50 pgg/ml). There was very little statistical variation between the effects of the two different types of LPS used. Due to the limited amount of A. actinomycetemcomitans LPS available for analysis, the remainder of the experiments was done with S. enteritidis LPS. Time-course studies to determine how quickly the stimulation of hyaluronate synthesis became effective in the presence of 50 pg/ml LPS showed a steady increase in the release of 3H-labelled hyaluronate into
the medium over 72 h for both treated and untreated cultures (Fig. 2). At all times, LPS-treated cells released more ‘H-labelled hyaluronate, but this difference was not statistically significant until 48 h (p < 0.05). The amount of radiolabelled hyaluronate associated with the cell layers of treated and untreated cultures also increased over time. Although the LPS-treated cells appeared to have greater amounts of 3H-labelled hyaluronate associated with their cell layers, there was little statistical difference between them even after 72 h. The effect of LPS on newly synthesized, cell-associated, [3H]-glucosamine-lablled hyaluronate, assessed after a pulse chase for various times (Fig. 3) was to
-i
;LLL)I 50 LPE; concentration @g/ml)
0
0.5
5
Fig. 1. Effect of LPS from an oral and non-oral source on the release of hyahtronate into the medium by human gingival fibroblasts. Triplicate cultures of human gingival fibroblasts were maintained in the presence or absence of various concentrations of 5’. enteritidis and A. actinomycetemcomitans LPS. The incorporation of [a]-glucosamine into hyaluronate was monitored after 24 h. Data are presented as the mean and SD of the mean from triplicate cultures. Abbreviations: Se., S. enteritidisLPS; A.a., A. acrinomycetemcomita LPS.
--I 0
20
40
60
60
h
Fig. 2. Time course of the effect of LPS on hyaluronate synthesis. Triplicate cultures of human gingival fibroblasts were maintained in the presence or absence of 50 pg/ml S. enteritidisLPS and the incorporation of [‘HI-glucosamine into hyaluronate was monitored over 48 h for both the medium and cell layer. Data are presented as the mean and SD of the mean.
P. M. BARTOLD
794
produce no remarkable differences in the kinetics of release of labelled hyaluronate between treated or untreated cells. The labelled hyaluronate associated with the cell layer initially decreased rapidly and then more slowly, reaching 50% of the zero-time value by around 15 h. This decrease was accompanied by a concomitant increase of labelled hyaluronate in the medium. As hyaluronate synthesis is regulated by the enzyme hyaluronate synthetase, the effect of LPS on this enzyme was monitored at various times after introduction of LPS to the cultures (Fig. 4). After isolation of the enzyme from the cells, incubation with the appropriate radioactive substrates showed increased hyaluronate synthetase activity at 48 h in the control cultures. There was greater enzyme activity at all times in those cultures exposed to LPS; by 48 h, these had an approx. 100% increase in activity, while the controls had a 60% increase. To determine whether the cell response was a direct effect of LPS, or mediated via a secondary signal such as increased PGE, production, endogenous PGE, synthesis was blocked by exposing the cells to indomethacin (Fig. 5). In the absence of indomethacin, hyaluronate synthesis was significantly greater in the cultures exposed to LPS than in controls. Although indomethacin did not appear to influence hyaluronate synthesis greatly in the controls, it re-
(a)
40
24
a
f LPS on hyaluronate synthetase acitivity at various ume points. Enzyme activity was assessed by the incorporation of UDP-D-[U-“C]-glucuronic acid into hyaluronate at selected time points in either the presence or absence of 50 pg/ml S. enteritidisLPS. Data represent the
mean and SD of the mean of triplicate cultures. duced hyaluronate synthesis in LPS-treated cultures to control levels. In the assays of effect of LPS on the molecular size of newly synthesized hyaluronate (Fig. 6), macromolecular material from both the cell layers and the medium eluted from Sepharose CL-2B in two major populations; one which was excluded from the gel and the other which eluted in the included volume. When these two broad molecular populations were digested with Streptomyces hyaluronidase and the hyaluronate content determined (Table 1), for the medium, most of the material in the void volume was hyaluronate, whereas a smaller proportion of the included peak was hyaluronate. Although a modest increase in larger molecular-weight hyaluronate was evident in the LPS-treated cultures, the differences were not significant, owing to the large variance. Similar findings were made for material isolated from the cell layers (Table 1). DISCUSSION
“I
0
lb
2-o
4’0
3’0
h
100
(b)
1
Crude extracts of human dental plaque can alter fibroblast proliferation, metabolism and hyaluronate synthesis (Larjava et al., 1983; Larjava, 1984; Larjava and Jalkanen, 1984). These observations are expanded by my initial findings that LPS, from two different sources, can influence hyaluronate synthesis
80 6040 20 -
O-
0
-LFS
20
Control
40
h
Fig. 3. Pulse chase of newly synthesized hyaluronate. Triplicate cultures of human gingival fibroblasts were maintained in the presence or absence of 50 pg/rnl S. enteritidis LPS and the incorporation of [‘HI-glucosamine into hyaluronate was allowed to proceed for 24 h. The medium was then removed and the cells washed extensively before incubation in isotope-free medium for various periods of time. The presence of [3H]-hyaluronatein both the cell layers (a) and medium (b) was determined by selective digestion with Streptomyces hyaluronidase. Data are presented as the mean and SD of the mean.
0
0.001
0.01
0.1
Indomethacln Concentration (mm) Fig. 5. Effect of inhibition of PGE, synthesis on hyaluronate synthesis. Triplicate cultures of human gingival fibroblasts were maintained in the presence or absence of 50 rg/ml S. enteritidisLPS and exposed to a range of concentrations of indomethacin known to inhibit prostaglandin synthesis by fibroblasts. Data are presented as the mean and SD of the mean.
795
LPS and fibroblast hyaluronate
by human gingival fibroblasts in vitro. Regardless of the source, LPS stimulated the amount of hyaluronate associated with cells that had been exposed to LPS. However, various LPS molecules are known to differ in their biological activity, their chemical composition depending upon (Kotani and Takada, 1990). Therefore, whether these initial observations reflect a general effect of all LPS molecules or are specific for certain types of LPS remains to be established. Because of the limited amount of LPS from A. actinomycetemcomitans available, I was able to study only the effects of S. enteritidis LPS on hyaluronate synthesis. The increase in macromolecular hyaluronate associated with cells exposed to LPS appears to be in direct contrast to previous findings that LPS inhibited synthesis of proteoglycans and proteins by human gingival fibroblasts (Bartold and Millar, 1988). Nonetheless, my present findings are in agreement with several other studies in which there was stimulation of hyaluronate synthesis by synovial and lung fibroblasts exposed to various concentrations of LPS from E. coli (Buckingham, Castor and Hoag, 1972; Buckingham and Castor, 1975). Furthermore, LPS has a &fferenti,al effect on prbteoglycan and Media 3000 I-,
-
ctrnlrd LFS
(4 4
g-2000-
E 3i B lOOO-
x 0
9 . 0
40
20
60
80
100
Fraction Number
Cell Layer
(b)
-w
~toooo-
i
Control
-LPS
E
2
‘D I
5000 -
0
Fraction Number
Fig. 6. Sepharose CL-:!B gel filtration chromatograms of 3H-labelled macromolecules isolated from (a) medium and (b) cell layer of human gingival fibroblast cultures maintained for 24 h in either the presence or absence of 50 pg/ml S. enteritidisLPS. The void volume (u,) and total volume (03 were determined by the elution of [3H]-DNA and [3sS]sulphate respectively an’d are marked with arrow heads. The bars represent the fractions which were pooled for subsequent hyaluronate determinations in different molecular size pools.
Table 1. Effect of LPS on molecular-sire distribution hyaluronate Control*
Media Cell layer
of
LPS
Peak 1
Peak 2
Peak 1
Peak 2
69* 17 45 f 9
24*4 7*t
82fll 49*5
IS&7 5*1
*Data are the percentage of 3H-labelled material digested by Streptomyceshyaluronidase.
hyaluronate synthesis et al., 1983).
by lung
fibroblasts
(Castor
The increase in the amount of labelled hyaluronate associated with cells exposed to LPS could reflect either an increase in the rate of synthesis or a decrease in rate of breakdown. From my results, it seems likely that the effect is one of increased synthesis. First, cells exposed to LPS incorporated [‘HI-glucosamine into hyaluronate faster than did control cells (time-course studies). Secondly, the release of newly labelled hyaluronate from cells exposed to LPS proceeded at a rate similar to that in the control cultures (pulsechase experiments). As the breakdown of glycosaminoglycans may be reflected in the rate of loss of newly labelled glycosaminoglycans from cells (Fratantoni, Hall and Neufeld, 1968; Hopwood and Dorfman, 1977) these findings indicate that the alteration in levels of hyaluronate associated with cells exposed to LPS represents an increase in the rate of synthesis as distinct from a decreased rate of breakdown. LPS at high concentrations is toxic to gingival fibroblasts (Bartold and Millar, 1988), so the response of increased hyaluronate synthesis by cells exposed to LPS may be the harbinger of toxicity. However, although LPS may exert a permissive effect on cells, enabling them to respond more efficiently to serum stimulants (Smith, 1976), the precise mechanism by which stimulation of hyaluronate synthesis occurs is not yet clear. Indeed, hyaluronate synthesis is not regulated in the same way as that of other known glycosaminoglycans (Kleine, 1981), and attempts to influence hyaluronate synthesis by chemicals that affect the synthesis of other glycosaminoglycans have been unsuccessful (Hart and Lennarz, 1978; Hopwood and Dorfman, 1977; Mapleson and Buchwald, 1981). One explanation for such findings may be that the site of hyaluronate synthesis seems to be at the plasma membrane rather than the Golgi (Prehm, 1984). Another explanation of the stimulation of hyaluronate synthesis may lie in the effect of LPS on the proliferation rate of the cells. LPS inhibits gingival fibroblast proliferation (Bartold and Millar, 1988); also, upon inhibition of gingival fibroblast proliferation with hydroxyurea, hyaluronate synthesis is increased (Bartold, 1988). This may reflect a response of the cells to being in a subconfluent state, as hyaluronate production is related not only to proliferation but also cell density (Hopwood and Dorfman, 1977; Toole, 1981). It is possible that a similar response to cell densities may apply when the cells are exposed to LPS, but all my experiments were done with confluent monolayer cultures to reduce such an effect.
796
P. M. BARTOLD
The level of hyaluronate synthetase activity was raised in cultures exposed to LPS. This increase was consistent with the observed stimulation of hyaluronate synthesis by LPS and supports the notion that the amount of hyaluronate production varied with time and exposure to LPS. Although an increase in hyaluronate synthetase activity with time has been reported before (Mian, 1986; Brecht et al., 1986), my data are interesting in that they show a cumulative effect of an endogenous agent on the activity of this enzyme. A further complicating factor in studies concerning the control of extracellular matrix production is the role of secondary mediators. One commonly encountered mediator is PGE,: PGE, synthesis by synovial fibroblasts is increased by LPS (Yaron et al., 1980); PGE, can stimulate the synthesis of hyaluronate by fibroblasts (Yaron et al., 1978). Therefore, the LPSinduced enhancement of hyaluronate synthesis could have been mediated through a PGE,-dependent pathway. I found that inhibition of PGE, synthesis by indomethacin abolished the stimulatory effect of LPS on hyaluronate synthesis. Thus the stimulation of hyaluronate by LPS may be secondary to induced endogenous PGE2 synthesis. My work confirms the findings of a previous study which showed selective size distribution of hyaluronate between the different cell-culture compartments of fibroblasts (Nakamura et al., 1990). My finding that the molecular size of the newly synthesized hyaluronate was not affected by LPS is important. In chronic periodontal disease, significant alterations occur in both the content and size of hyaluronate (Bartold and Page, 1986b). Whether these changes are the result of exogenous factors (e.g. enzymes and superoxides) or because of altered cell metabolism has been unclear. Although my study does not completely resolve this question, the findings imply that LPS does not contribute directly to the alteration in the molecular size of hyaluronate found in chronic gingival inflammation. Hyaluronate is considered to play a significant part in the regulation and maintenance of normal tissue functions (Comper and Laurent, 1978). For example, it plays a primary role during wound repair and can significantly influence macrophage, lymphocyte and fibroblast functions (Balazs and Darzynkiewicz, 1973; Forrestor and Balazs, 1980; Goldberg and Toole, 1987). That LPS stimulates hyaluronate synthesis by human gingival fibroblasts in vitro suggests that this could be a fundamental response by the cells to produce an extracellular matrix that is conducive to adequate host defence and repair. Acknowledgements-This study was supported by a grant from the National Health and Medical Research Council of Australia. The generous gift of LPS from A. actinomycetemcomitans, prepared by Dr S. J. Millar (Dr E. Hausman’s Laboratory), State University of New York at Buffalo is gratefully acknowledged. I also thank Miss M. Weger for her fine technical assistance. REFERENCES Appel A., Horwitz A. L. and Dorfman A. (1979) Cell-free synthesis of hyaluronic acid in Marfan syndrome. J. biol. Chem. 2!34, 12,199-12,203.
Balazs E. A. and Darzynkiewicz Z. (1973) The effect of hyaluronic acid on fibroblasts, mononuclear phagocytes and lymphocytes. In Biology of the Fibroblast (Eds Kulonen E. and Pikarainen J.), pp. 237-252. Plenum Press, New York. Bartold P. M. (1987) Proteoglycans of the periodontium: structure, role and functions. J. periodont. Rex 22, 431-444. Bartold P. M. (1988) The effect of interleukin-lb on hyaluronic acid synthesis by adult human gingival fibroblasts in vitro. J. periodont. Res. 23, 139-147. Bartold P. M. and Millar S. J. (1988) Effect of lipopolysaccharide on proteoglycan synthesis by adult human gingival fibroblasts in vitro. Infect. Immun. 56, 2149-2155. Bartold P. M. and Page R. C. (1986a) Hyaluronic acid synthesized by fibroblasts from normal and chronically inflamed gingiva. Collagen rel. Rex 6, 365-377. Bartold P. M. and Page R. C. (1986b) The effect of chronic inflammation on soft connective tissue proteoglycans and hyaluronic acid. J. oral Path. 15, 367-374. Brecht M., Mayer U., Schlosser E. and Prehm P. (1986) Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem. J. 239, 445-450. Buckingham R. B. and Castor C. W. (1975) Rheumatoid behaviour in normal human synovial fibroblasts induced by extracts of Gram-negative bacteria. J. Lab. clin. Med. 85, 422-435. Buckingham R. B., Castor C. W. and Hoag P. F. (1972) The effect of bacterial products on synovial fibroblast function: Hypermetabolic changes induced by endotoxin. J. clin. Invest. 51, 1186-l 194. Castor C. W., Bignall M. C., Hossler P. A. and Roberts D. J. (1981) Connective tissue activation-XXl. Regulation of glycosaminoglycan metabolism by lymphocyte (CTAP-I) and platelet (CTAP-III) growth factors. In Vitro 17, 777-785. Castor C. W., Fremuth T. D., Furley A. M. and Jourdian G. W. (1983) Hvaluronic acid and nroteoalvcan synthesis by lung fib&blasts in basal and aciivatedsiates. in Vitro 19, 462-470. Comper W. D. and Laurent T. C. (1978) Physiological function of connective tissue polysaccharides. Physiol. Rev. 58, 255-315. Daly C. G., Seymour G. J. and Kaiser J. B. (1980) Bacterial endotoxin: a role in chronic inflammatory periodontal disease? J. oral Path. 9, l-15. Dayer J.-M., Goldring S. R., Robinson D. R. and Krane S. M. (1979) Effects of human mononuclear cell factor on cultured rheumatoid synovial cells. Biochim. biophys. Acta 586, 87-105. Forrester J. V. and Balazs E. A. (1980) Inhibition of phagocytosis by high molecular weight hyaluronate. Immunology 40, 43545. Fratantoni J. C., Hall C. W. and Neufeld E. F. (1968) The defect in the Hurler’s and Hunter’s syndromes: faulty degradation of mucopolysaccharides. Proc. natn. Acad. Sci. U.S.A. 60, 699-706. Goldberg R. L. and Toole B. P. (1987) Hyaluronate inhibition of cell proliferation. Arthritis Rheum. 30. 769-778. Hanazawa S., Nakada K., Ohmori Y., Miyoshi T., Amano S. and Kitano S. (1985) Functional role of interleukin-1 in periodontal disease: induction of interleukin-1 production by Bacteroides gingivalis lipopolysaccharide in neritoneal macronhaaes from C3H/HeN and C3H/HeJ mice. Infect. Immk-9, 62-270. Hart G. W. and Lennarz W. J. (1978) Effects of Tunicamycin on the biosynthesis of glycosaminoglycans by embryonic chick cornea. J. biol. Chem. 253, 5795-5801. Hausman E.. Raisz L. G. and Miller W. J. (1970) Endotoxin stimulation of bone resorption in tissue culture. Science 168,862-864.
LPS and fibroblast hyaluronate Hopwood J. J. and Dorfman A. (1977) Glycosaminoglycans synthesized by cultured human skin fibroblasts after transformation with Simian virus 40. J. biof. Chem. 252, 4111-4185. Huey G., Stair S. and Stem R. (1990) Hyaluronic acid determinations: optimizing assay parameters. Matrix 10, 61-74. Kleine T. 0. (1981) Biosynthesis of proteoglycans: an approach to locate it in different membrane systems. ht. Rev. Connect. Tiss. Rev. 9, 21-98.
Kotani S. and Takada H. (1990) Structural requirements of lipid A for endotoxicity and other biological activitiesan overview. Adv. expl Biol. Med. 256, 1343. Larjava H. (1984) Metabolic change in cultured gingival fibroblasts exposed to bacterial extracts. Stimulation of hyaluronic acid synthesis. J. periodont. Res. 19, 230-237.
Larjava H. and Jalkanen M. (1984) Cell surface glycoconjugates of gingival fibroblasts exposed to dental plaque extract. J. periodont. Res. 19, 469-482. Larjava H., Jalkanen M., Pentinen R. and Paunio K. (1983) Enhanced synthesis of hyaluronic acid by human gingival fibroblasts exposed to human dental bacterial plaque. J. periodont. Res. 18, 31-39. Larjava H., Uitto V.-J., Eerola E. and Haaspasalo M. (1987) Inhibition of gingival fibroblast growth by Bacieroides gingivalis. In,&ci Immun. 55, 210-205. Manleson J. L. and Buchwald M. (1981) Effect of cvclohex&de and dexametha.sone phosphate’ on hyaluronic acid synthesis and secretion in cultured human skin fibroblasts. J. Cell Physiol. 109, 215-222. Mian N. (1986) Analysis of cell-growth-phase-related variations in hyaluronate synthetase activity of isolated plasma-membrane fractions of cultured human skin fibroblasts. Biochem. J. 231, 333-342. Millar S. J., Goldstein E. G., Levine M. J. and Hausman E. (1986) Modulation of bone metabolism by two chemically distinct lipopolysaccharide fractions from Bacteroides gingivalis. Infect. Immun. 51, 302-306. Nakamura T., Takagaki K., Kubo K., Morikawa A., Tamura S. and Endo M. (1990) Extracellular depolymer-
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ixation of hyaluronic acid in cultured human skin fibroblasts. Biochem. biophys. Res. Commun. 172, 70-76. Narayanan A. S. and Page R. C. (1983) Connective tissues of the periodontium: a summary of current work. Collagen rel. Res. 3, 33-64. Narayanan A. S., Page R. C. and Kuxan F. (1978) Collagens svnthesized in vitro bv dinloid fibroblasts obtained from chronically inflamed human connective tissue. Lab. Invest. 39, 61-65. Page R. C. and Schroeder H. E. (1976) Pathogenesis of inflammatory periodontal disease. A summary of current work. Lab. Invest. 33, 235-246. Prehm P. (1984) Hyaluronate is synthesized at plasma membranes. Biochem. J. 2M, 597-600. Smith G. L. (1976) Synergistic action of bacterial lipopolysaccharides on serum stimulated DNA synthesis in mouse embryo fibroblasts. Proc. Sot. exp. Biol. Med. 153, 187-192.
Toole B. P. (1981) Glycosaminoglycans in morphogenesis. In Cell Biology of the Extracellular Matrix (Ed. Hay E. D.), pp. 259-294. Plenum Press, New York.. Vaheri A., Ruoslahti E., Sarvas M. and Nurminen M. (1973) Mitogenic effect by lipopolysaccharide and pokeweed mitogen on density-inhibited chick embryo fibroblasts. J. exp. Med. 138, 13561364. Wahl L. M., &en C. E., Sandberg A. L. and Mergenhagen S. E. (1977) Prostaglandin regulation of macrophage collagenase production. Proc. natn. Acad. Sci. U.S.A. 14, 49554958.
Westphal 0. and Jann K. (1965) Bacterial lipopolysaccharide extraction with phenol water and further application of these procedures. Methodr Carbohydr. Chem. 5, 83-91. Yaron M., Yaron I., Wiletxki C. and Zor U. (1978) Interrelationship between stimulation of prostaglandin E and hyaluronate production by Poly (I), Poly (C) and interferon in synovial fibroblast culture. Arthritb Rheum. 21, 694698.
Yaron M., Yaron I., Smetana O., Eylan E. and Zor U. (1980) Stimulation of prostaglandin E production by bacterial endotoxins in cultured human synovial fibroblasts. Arthritis Rhewn. 23, 921-925.
ooo3-9969/91 S3.00 + 0.00 Copyright 0 1991 Pcrgamon Press plc
LIPOPOLYSACCHARIDE STIMULATION OF HYALURONATE SYNTHESIS BY HUMAN GINGIVAL FIBROBLASTS IN VITRO P. M. BARTOLD Department of Pathology, University of Adelaide, Adelaide, Australia (Accepted 31 May 1991) Summary-Exposure of gingival fibroblasts to LPS caused a dose-dependent increase in hyaluronate synthesis. Stimulation of hyaluronate synthesis by LPS was significantly greater 24 h after exposure and by 48 h an approx. 50% increase was evident. In parallel, there was an increase in the activity of the hyaluronate synthetase enzyme. Inhibition of PGE, synthesis by indomethacin abolished the stimulatory effect of LPS on hyaluronate synthesis. Thus, this stimulatory effect of LPS on hyaluronic acid synthesis may be a secondary response to the induction of PGE,. The molecular size of newly synthesized hyaluronate was not affected by LPS. The metabolic changes observed may be a primary response of the cells to bacterial toxins and may aid extracellular matrix repair. Key words: hpopolysaccharide,
hyaluronate, fibroblasts.
INTRODUCTION Lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria, has received considerable attention in relation to the pathogenesis of the periodontal diseases (Daly, Seymour and Kieser, 1980). In particular, it has been shown to
modulate cell growth (Vaheri et al., 1973; Larjava et al., 1987), stimulate bone resorption (Hausman, Raisz and Miller, 1970; Millar et al., 1986) and induce lymphokine release by macrophages (Hanazawa et al., 1985). Thus, it is considered to be a potent biological agent capable of important effects in infected tissues either by direct influences on cell metabolism, or, indirectly, by invoking an inflammatory response. The connective tissues of the periodontium have both fibrous and non-fibrous elements which, together, impart structural and physiological properties that allow adequate function (Narayanan and Page, 1983; Bartold, 1987). However, upon induction of inflammation, numerous changes occur within the extracellular matric.es of the periodontium (Page and Schroeder, 1976). In addition to ultrastructural changes, the metabolism of the resident fibroblasts is also disturbed. The combination of tissue destruction and altered cellular activity leads to compromised function. In recent years, the metabolism and degradation of periodontal collagens has been well studied (Narayanan and Page, 1983). However, other principal components of the extracellular matrix such as proteoglycans and hyaluronate have received less attention (Bartold, 1987). Therefore, as hyaluronate has been implicated in critical functions such as the Abbreviarions: CPC’, cetylpyridinium chloride; DVM, Dulbecco-Vogt medium; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; PG, prostaglandin.
regulation of cell-cell interactions, tissue hydration, and control of macrophages and lymphocytes (Comper and Laurent, 1978), my purpose now was to investigate the effect of LPS on hyaluronate synthesis by human gingival fibroblasts in vitro. MATERIALS
AND METHODS
Materials
Guanidine HCl, indomethacin, glutamine, adenosine 5’ triphosphate (ATP), and UDP-N-acetylglucosamine were all purchased from the Sigma Chemical Co., St Louis, MO, U.S.A. CPC was from Ajax Chemicals Australia, Aulam, N.S.W.; filter paper (3MM) from Whatman, Maidstone, U.K.; Streptomyces hyaluronidase from Seikagaku Kogyo Ltd, Tokyo, Japan; Streptomyces griseus pronase from Calbiochem, San Diego, CA; Sephadex G-50 and Sepharose CL-2B from Pharmacia Fine Chemicals, North Ryde, Sydney, Australia; DVM, fetal bovine serum, penicillin, streptomycin and nonessential amino acids were all from Flow Laboratories, Irvine, Ayrshire, U.K.; tissue-culture ware was from Nunc, Roskilde, Denmark; o-[6-3H]-g1ucosamine HCl(33 Ci/mmol) and UDP-D-[U-14C]-glucuronic acid (225 mCi/mmol) were from Amersham (Australia), Sydney, Australia; Ready Solv EP scintillation fluid was from Beckman (Australia), Adelaide, Australia. LPS preparation
LPS from Actinobacillus actinomycetemcomitans was a gift from Dr E. Hausman’s laboratory, State University of New York at Buffalo and had been extracted by the phenol-water method (Westphal and Jann, 1965). Although no subsequent purification was carried out, and it was considered a crude LPS preparation, it has significant biological activity 791
792
P.M. BARTOLD
attributable to LPS (Bartold and Millar, 1988). LPS from Salmonella enteritidis was purchased from Sigma Chemical Co., St Louis, MO, U.S.A. It had been extracted with phenol and purified by gel filtration chromatography. Both its protein and RNA contents were less than 1%. Fibroblast cultures Fibroblasts were obtained by explant culture from biopsies of healthy human gingiva from volunteers, as described by Narayanan, Page and Kuzan (1978); these have been used extensively in previous studies (Bartold and Page, 1986a; Bartold and Millar, 1988). The cells were maintained in DVM supplemented with 10% heat-inactivated fetal bovine serum, 100 units/ml of penicillin, 100 pg/ml streptomycin, 2 mM glutamine and non-essential amino acids at 37°C in an atmosphere of 5% CO, and 95% air. Cells between the 4th and 8th transfer in culture were used. All experiments, unless otherwise stated, were carried out in complete DVM containing 10% fetal bovine serum. Radioisotope incorporation studies Initial studies were designed to investigate the effect of various preparations of LPS on hyaluronate synthesis by the fibroblasts. Fibroblasts were seeded in triplicate at a concentration of 5 x lo4 cells per well, into 24-well plates. Upon reaching confluence (1 x lo5 cells/well), the cells were incubated in medium containing LPS from either S. enteritidis or A. actinomycetemcomitans ranging from 50 to 0.5 pg/ml for 24 h and pulsed with [3H]-glucosamine (20 pCi/ml) for the entire period. The medium was then removed and the cell layers washed once with PBS, which was pooled with the medium to make up a fraction for subsequent analyses of hyaluronate content. The cells were released from the culture plates by trypsinization. A sample was taken for counting cell numbers in a haemocytometer and the remainder was frozen until required. To determine the effect of LPS on the rate of hyaluronate synthesis, confluent cells were cultured as described above. Upon reaching confluence the medium was removed and the cells incubated in the presence or absence of 50pg/ml S. enteritidis LPS for 4, 8, 24, 30 48 and 12 h. The incorporation of [‘HI-glucosamine into hyaluronate was determined by selective digestion with hyaluronidase for each time point after the introduction of the radiolabel and 50 pg/ml LPS. As PGE? can influence hyaluronate synthesis and because LPS has been reported to stimulate prostaglandin synthesis in other systems (Yaron et al., 1978; Wahl et al., 1977) the effect of S. enteritidis LPS on hyaluronate synthesis in the absence of endogenous PGE, was investigated. Gingival fibroblasts were grown to confluence in 24-well plates and incubated in the presence or absence of 50 pg/ml LPS in DVM containing 20 pCi/ml [3H]-glucosamine for 24 h. To inhibit PGE, synthesis, indomethacin ranging in concentration from 10m6 to 10m4M was also added to some cultures. This concentration range inhibited PGE, synthesis by fibroblasts in culture (Dayer et al., 1979) and was without toxic effects on human
gingival fibroblasts (Bartold, 1988). Hyaluronate synthesis was monitored by hyaluronidase digestion of molecules labelled with [3H]-glucosamine. To obtain material for molecular-size analysis, human gingival fibroblasts were grown to confluence in 25-cm* culture flasks. Upon attaining confluence (5 x lo5 cells per flask) the culture medium was removed and the cells cultured in the presence or absence of 5Opg/ml S. enteritidis LPS for 24 h in DVM containing 20 pC!i/ml [‘HI-glucosamine. After incubation the medium was removed and the cell layers washed with 3 ml PBS which was then pooled with the medium to make up this fraction. The remaining cells in culture vessels were then extracted with 5 ml of 4 M guanidine HCl/O.OS M sodium acetate, pH 5.8, overnight at 4°C. After 24 h at 4°C this cell fraction was centrifuged to remove any debris and the supematant used for subsequent analyses as the cell-layer fraction. Both the medium and cell-layer extracts were desalted by gel filtration chromatography on Sephadex G-50 to remove unincorporated [3H]-glucosamine (Bartold and Page, 1986a). The labelled macromolecular material which eluted in the void volume of Sephadex G-50 (1 x 30 cm) was used for subsequent analyses of hyaluronate molecular size. Determination of 3H-labelled hyaluronate Hyaluronate synthesis by human gingival fibroblasts in vitro was determined as described previously (Castor et al., 1981; Huey, Stair and Stem, 1990). In brief, samples (200 ~1) from the culture medium were digested with pronase (10 mg/ml) overnight at 60°C. They were then boiled and split into two portions, each of 50 ~1. One portion (designated B) was incubated at 37°C for 12 h with 60~1 Streptomyces hyaluronidase in 0.1 M sodium acetate buffer at pH 6.0. The other (designated A) was incubated in the sodium acetate buffer alone under identical conditions. After the incubation, samples (100 ~1) from each part were spotted onto filter paper divided into 2-cm squares, allowed to dry at 60°C and then placed through four washes, each of 30-min duration, in 0.05 M sodium chloride containing 0.1% CPC at room temperature (22°C). The filter paper was then removed and allowed to dry. Each square was then cut into small strips and placed in 14ml of scintillation fluid before determining the radioactivity in a liquid scintillation counter. The amount of hyaluronate in each sample was calculated as the amount of radioactivity digested by the Streptomyces hyaluronidase (i.e. dis/min in portion A - dis/min in portion B. Pulse-chase experiments To determine whether or not the effect of LPS on hyaluronate synthesis was due to an increase in rate of synthesis or decrease in rate of breakdown, pulsechase experiments were made. Fibroblasts were seeded, in triplicate at an initial density of 5 x lo4 cells per well, and allowed to grow to confluency (1 x 10’ cells/well). The confluent monolayers were then incubated in the presence of 2Opg/ml [‘HIglucosamine and 50/rg/ml LPS for 24 h, washed four times with PBS and then chased in 500~1 of
793
LPS and fibroblast hyaluronate
isotope-free medium containing 50 pg/ml LPS for various times. At each time, the medium was removed and the cell layers scraped from the plates. Hyaluronic determinations were carried out on both the medium and &-layer fractions. Hyah4ronate synthetase activity
Molecular-sieve chromatography
The molecular size of hyaluronate synthesized in the presence or absence of S. enteritidis LPS was assessed after 48 h in culture using Sepharose CL-ZB, as described by Bartold and Page (1986a). Approximately the same am’ounts (40,000 dis/min) of radiolabelled macromolecular material isolated from the medium and cell-layer fractions by Sephadex G-50 chromatography were eluted from Sepharose CL-2B (0.7 x 100 cm) with 4 M guanidine HCl in 0.05 M sodium acetate, pH 5.8. With a flow rate of 3 ml/h, fractions of 0.5 ml were collected and 50-~1 samples were taken for radioactivity determination in a liquid scintillation counter. The material which eluted in the void volume was pooled separately from that which eluted in the included volume. These two pools were indicative of the two major molecular-size fractions of [3H]-glucosamine-labelled material eluting from Sepharose CL-2B. After dialysis against 0.1 M
c I m
-
0
Statistical analysis
All data were subjected to analysis of variance.
Gingival fibroblasts were grown to confluence in 24-well plates and cultured in the presence or absence of 50 pg/ml S. enteritidis LPS for 8,24 and 48 h. The cells were removed from the culture surface by trypsinization and the cell pellet obtained by gentle centrifugation was washed twice with PBS before freezing at -70°C. The cells were lysed by freezing and thawing the pellet three times. Hyaluronate synthetase activity was determined by incubating the lysed cells in the presence of 0.1 PCi UDP-D-[U-‘~C]glucuronic acid and 5 x 10-3 PM UDP-N-acetylglucosamine as substrates (Appel, Horwitz and Dorfman, 1979). The: reaction mixture was incubated for 90 min at 37°C and the amount of hyaluronate thus synthesized was determined by selective digestion with hyaluronidase as described above.
3000 = .I-_E .$2000 =In 8 m0 1000
sodium acetate, the hyaluronate content in these fractions was determined by selective digestion with Streptomyces hyaluronidase (Bartold and Page, 1986a).
m Control q se. n A.&
RESULTS The initial experiments to determine the biological effect of LPS on hyaluronic acid synthesis (Fig. l), showed that, in all cases, exposure of the fibroblasts to higher (5 and SOpg/ml) concentrations of LPS increased that synthesis. Concentrations of LPS up to 50 pg/ml had no visible effect on the cells whereas at concentrations of 500 pg/ml, detachment and death occurred (data not shown). Cell counts after the experiments showed no significant change between LPS-treated and -untreated cells (data not shown). Regardless of the source of the LPS, the greatest stimulation of hyaluronate production occurred at the highest concentration which did not cause cell death (50 pgg/ml). There was very little statistical variation between the effects of the two different types of LPS used. Due to the limited amount of A. actinomycetemcomitans LPS available for analysis, the remainder of the experiments was done with S. enteritidis LPS. Time-course studies to determine how quickly the stimulation of hyaluronate synthesis became effective in the presence of 50 pg/ml LPS showed a steady increase in the release of 3H-labelled hyaluronate into
the medium over 72 h for both treated and untreated cultures (Fig. 2). At all times, LPS-treated cells released more ‘H-labelled hyaluronate, but this difference was not statistically significant until 48 h (p < 0.05). The amount of radiolabelled hyaluronate associated with the cell layers of treated and untreated cultures also increased over time. Although the LPS-treated cells appeared to have greater amounts of 3H-labelled hyaluronate associated with their cell layers, there was little statistical difference between them even after 72 h. The effect of LPS on newly synthesized, cell-associated, [3H]-glucosamine-lablled hyaluronate, assessed after a pulse chase for various times (Fig. 3) was to
-i
;LLL)I 50 LPE; concentration @g/ml)
0
0.5
5
Fig. 1. Effect of LPS from an oral and non-oral source on the release of hyahtronate into the medium by human gingival fibroblasts. Triplicate cultures of human gingival fibroblasts were maintained in the presence or absence of various concentrations of 5’. enteritidis and A. actinomycetemcomitans LPS. The incorporation of [a]-glucosamine into hyaluronate was monitored after 24 h. Data are presented as the mean and SD of the mean from triplicate cultures. Abbreviations: Se., S. enteritidisLPS; A.a., A. acrinomycetemcomita LPS.
--I 0
20
40
60
60
h
Fig. 2. Time course of the effect of LPS on hyaluronate synthesis. Triplicate cultures of human gingival fibroblasts were maintained in the presence or absence of 50 pg/ml S. enteritidisLPS and the incorporation of [‘HI-glucosamine into hyaluronate was monitored over 48 h for both the medium and cell layer. Data are presented as the mean and SD of the mean.
P. M. BARTOLD
794
produce no remarkable differences in the kinetics of release of labelled hyaluronate between treated or untreated cells. The labelled hyaluronate associated with the cell layer initially decreased rapidly and then more slowly, reaching 50% of the zero-time value by around 15 h. This decrease was accompanied by a concomitant increase of labelled hyaluronate in the medium. As hyaluronate synthesis is regulated by the enzyme hyaluronate synthetase, the effect of LPS on this enzyme was monitored at various times after introduction of LPS to the cultures (Fig. 4). After isolation of the enzyme from the cells, incubation with the appropriate radioactive substrates showed increased hyaluronate synthetase activity at 48 h in the control cultures. There was greater enzyme activity at all times in those cultures exposed to LPS; by 48 h, these had an approx. 100% increase in activity, while the controls had a 60% increase. To determine whether the cell response was a direct effect of LPS, or mediated via a secondary signal such as increased PGE, production, endogenous PGE, synthesis was blocked by exposing the cells to indomethacin (Fig. 5). In the absence of indomethacin, hyaluronate synthesis was significantly greater in the cultures exposed to LPS than in controls. Although indomethacin did not appear to influence hyaluronate synthesis greatly in the controls, it re-
(a)
40
24
a
f LPS on hyaluronate synthetase acitivity at various ume points. Enzyme activity was assessed by the incorporation of UDP-D-[U-“C]-glucuronic acid into hyaluronate at selected time points in either the presence or absence of 50 pg/ml S. enteritidisLPS. Data represent the
mean and SD of the mean of triplicate cultures. duced hyaluronate synthesis in LPS-treated cultures to control levels. In the assays of effect of LPS on the molecular size of newly synthesized hyaluronate (Fig. 6), macromolecular material from both the cell layers and the medium eluted from Sepharose CL-2B in two major populations; one which was excluded from the gel and the other which eluted in the included volume. When these two broad molecular populations were digested with Streptomyces hyaluronidase and the hyaluronate content determined (Table 1), for the medium, most of the material in the void volume was hyaluronate, whereas a smaller proportion of the included peak was hyaluronate. Although a modest increase in larger molecular-weight hyaluronate was evident in the LPS-treated cultures, the differences were not significant, owing to the large variance. Similar findings were made for material isolated from the cell layers (Table 1). DISCUSSION
“I
0
lb
2-o
4’0
3’0
h
100
(b)
1
Crude extracts of human dental plaque can alter fibroblast proliferation, metabolism and hyaluronate synthesis (Larjava et al., 1983; Larjava, 1984; Larjava and Jalkanen, 1984). These observations are expanded by my initial findings that LPS, from two different sources, can influence hyaluronate synthesis
80 6040 20 -
O-
0
-LFS
20
Control
40
h
Fig. 3. Pulse chase of newly synthesized hyaluronate. Triplicate cultures of human gingival fibroblasts were maintained in the presence or absence of 50 pg/rnl S. enteritidis LPS and the incorporation of [‘HI-glucosamine into hyaluronate was allowed to proceed for 24 h. The medium was then removed and the cells washed extensively before incubation in isotope-free medium for various periods of time. The presence of [3H]-hyaluronatein both the cell layers (a) and medium (b) was determined by selective digestion with Streptomyces hyaluronidase. Data are presented as the mean and SD of the mean.
0
0.001
0.01
0.1
Indomethacln Concentration (mm) Fig. 5. Effect of inhibition of PGE, synthesis on hyaluronate synthesis. Triplicate cultures of human gingival fibroblasts were maintained in the presence or absence of 50 rg/ml S. enteritidisLPS and exposed to a range of concentrations of indomethacin known to inhibit prostaglandin synthesis by fibroblasts. Data are presented as the mean and SD of the mean.
795
LPS and fibroblast hyaluronate
by human gingival fibroblasts in vitro. Regardless of the source, LPS stimulated the amount of hyaluronate associated with cells that had been exposed to LPS. However, various LPS molecules are known to differ in their biological activity, their chemical composition depending upon (Kotani and Takada, 1990). Therefore, whether these initial observations reflect a general effect of all LPS molecules or are specific for certain types of LPS remains to be established. Because of the limited amount of LPS from A. actinomycetemcomitans available, I was able to study only the effects of S. enteritidis LPS on hyaluronate synthesis. The increase in macromolecular hyaluronate associated with cells exposed to LPS appears to be in direct contrast to previous findings that LPS inhibited synthesis of proteoglycans and proteins by human gingival fibroblasts (Bartold and Millar, 1988). Nonetheless, my present findings are in agreement with several other studies in which there was stimulation of hyaluronate synthesis by synovial and lung fibroblasts exposed to various concentrations of LPS from E. coli (Buckingham, Castor and Hoag, 1972; Buckingham and Castor, 1975). Furthermore, LPS has a &fferenti,al effect on prbteoglycan and Media 3000 I-,
-
ctrnlrd LFS
(4 4
g-2000-
E 3i B lOOO-
x 0
9 . 0
40
20
60
80
100
Fraction Number
Cell Layer
(b)
-w
~toooo-
i
Control
-LPS
E
2
‘D I
5000 -
0
Fraction Number
Fig. 6. Sepharose CL-:!B gel filtration chromatograms of 3H-labelled macromolecules isolated from (a) medium and (b) cell layer of human gingival fibroblast cultures maintained for 24 h in either the presence or absence of 50 pg/ml S. enteritidisLPS. The void volume (u,) and total volume (03 were determined by the elution of [3H]-DNA and [3sS]sulphate respectively an’d are marked with arrow heads. The bars represent the fractions which were pooled for subsequent hyaluronate determinations in different molecular size pools.
Table 1. Effect of LPS on molecular-sire distribution hyaluronate Control*
Media Cell layer
of
LPS
Peak 1
Peak 2
Peak 1
Peak 2
69* 17 45 f 9
24*4 7*t
82fll 49*5
IS&7 5*1
*Data are the percentage of 3H-labelled material digested by Streptomyceshyaluronidase.
hyaluronate synthesis et al., 1983).
by lung
fibroblasts
(Castor
The increase in the amount of labelled hyaluronate associated with cells exposed to LPS could reflect either an increase in the rate of synthesis or a decrease in rate of breakdown. From my results, it seems likely that the effect is one of increased synthesis. First, cells exposed to LPS incorporated [‘HI-glucosamine into hyaluronate faster than did control cells (time-course studies). Secondly, the release of newly labelled hyaluronate from cells exposed to LPS proceeded at a rate similar to that in the control cultures (pulsechase experiments). As the breakdown of glycosaminoglycans may be reflected in the rate of loss of newly labelled glycosaminoglycans from cells (Fratantoni, Hall and Neufeld, 1968; Hopwood and Dorfman, 1977) these findings indicate that the alteration in levels of hyaluronate associated with cells exposed to LPS represents an increase in the rate of synthesis as distinct from a decreased rate of breakdown. LPS at high concentrations is toxic to gingival fibroblasts (Bartold and Millar, 1988), so the response of increased hyaluronate synthesis by cells exposed to LPS may be the harbinger of toxicity. However, although LPS may exert a permissive effect on cells, enabling them to respond more efficiently to serum stimulants (Smith, 1976), the precise mechanism by which stimulation of hyaluronate synthesis occurs is not yet clear. Indeed, hyaluronate synthesis is not regulated in the same way as that of other known glycosaminoglycans (Kleine, 1981), and attempts to influence hyaluronate synthesis by chemicals that affect the synthesis of other glycosaminoglycans have been unsuccessful (Hart and Lennarz, 1978; Hopwood and Dorfman, 1977; Mapleson and Buchwald, 1981). One explanation for such findings may be that the site of hyaluronate synthesis seems to be at the plasma membrane rather than the Golgi (Prehm, 1984). Another explanation of the stimulation of hyaluronate synthesis may lie in the effect of LPS on the proliferation rate of the cells. LPS inhibits gingival fibroblast proliferation (Bartold and Millar, 1988); also, upon inhibition of gingival fibroblast proliferation with hydroxyurea, hyaluronate synthesis is increased (Bartold, 1988). This may reflect a response of the cells to being in a subconfluent state, as hyaluronate production is related not only to proliferation but also cell density (Hopwood and Dorfman, 1977; Toole, 1981). It is possible that a similar response to cell densities may apply when the cells are exposed to LPS, but all my experiments were done with confluent monolayer cultures to reduce such an effect.
796
P. M. BARTOLD
The level of hyaluronate synthetase activity was raised in cultures exposed to LPS. This increase was consistent with the observed stimulation of hyaluronate synthesis by LPS and supports the notion that the amount of hyaluronate production varied with time and exposure to LPS. Although an increase in hyaluronate synthetase activity with time has been reported before (Mian, 1986; Brecht et al., 1986), my data are interesting in that they show a cumulative effect of an endogenous agent on the activity of this enzyme. A further complicating factor in studies concerning the control of extracellular matrix production is the role of secondary mediators. One commonly encountered mediator is PGE,: PGE, synthesis by synovial fibroblasts is increased by LPS (Yaron et al., 1980); PGE, can stimulate the synthesis of hyaluronate by fibroblasts (Yaron et al., 1978). Therefore, the LPSinduced enhancement of hyaluronate synthesis could have been mediated through a PGE,-dependent pathway. I found that inhibition of PGE, synthesis by indomethacin abolished the stimulatory effect of LPS on hyaluronate synthesis. Thus the stimulation of hyaluronate by LPS may be secondary to induced endogenous PGE2 synthesis. My work confirms the findings of a previous study which showed selective size distribution of hyaluronate between the different cell-culture compartments of fibroblasts (Nakamura et al., 1990). My finding that the molecular size of the newly synthesized hyaluronate was not affected by LPS is important. In chronic periodontal disease, significant alterations occur in both the content and size of hyaluronate (Bartold and Page, 1986b). Whether these changes are the result of exogenous factors (e.g. enzymes and superoxides) or because of altered cell metabolism has been unclear. Although my study does not completely resolve this question, the findings imply that LPS does not contribute directly to the alteration in the molecular size of hyaluronate found in chronic gingival inflammation. Hyaluronate is considered to play a significant part in the regulation and maintenance of normal tissue functions (Comper and Laurent, 1978). For example, it plays a primary role during wound repair and can significantly influence macrophage, lymphocyte and fibroblast functions (Balazs and Darzynkiewicz, 1973; Forrestor and Balazs, 1980; Goldberg and Toole, 1987). That LPS stimulates hyaluronate synthesis by human gingival fibroblasts in vitro suggests that this could be a fundamental response by the cells to produce an extracellular matrix that is conducive to adequate host defence and repair. Acknowledgements-This study was supported by a grant from the National Health and Medical Research Council of Australia. The generous gift of LPS from A. actinomycetemcomitans, prepared by Dr S. J. Millar (Dr E. Hausman’s Laboratory), State University of New York at Buffalo is gratefully acknowledged. I also thank Miss M. Weger for her fine technical assistance. REFERENCES Appel A., Horwitz A. L. and Dorfman A. (1979) Cell-free synthesis of hyaluronic acid in Marfan syndrome. J. biol. Chem. 2!34, 12,199-12,203.
Balazs E. A. and Darzynkiewicz Z. (1973) The effect of hyaluronic acid on fibroblasts, mononuclear phagocytes and lymphocytes. In Biology of the Fibroblast (Eds Kulonen E. and Pikarainen J.), pp. 237-252. Plenum Press, New York. Bartold P. M. (1987) Proteoglycans of the periodontium: structure, role and functions. J. periodont. Rex 22, 431-444. Bartold P. M. (1988) The effect of interleukin-lb on hyaluronic acid synthesis by adult human gingival fibroblasts in vitro. J. periodont. Res. 23, 139-147. Bartold P. M. and Millar S. J. (1988) Effect of lipopolysaccharide on proteoglycan synthesis by adult human gingival fibroblasts in vitro. Infect. Immun. 56, 2149-2155. Bartold P. M. and Page R. C. (1986a) Hyaluronic acid synthesized by fibroblasts from normal and chronically inflamed gingiva. Collagen rel. Rex 6, 365-377. Bartold P. M. and Page R. C. (1986b) The effect of chronic inflammation on soft connective tissue proteoglycans and hyaluronic acid. J. oral Path. 15, 367-374. Brecht M., Mayer U., Schlosser E. and Prehm P. (1986) Increased hyaluronate synthesis is required for fibroblast detachment and mitosis. Biochem. J. 239, 445-450. Buckingham R. B. and Castor C. W. (1975) Rheumatoid behaviour in normal human synovial fibroblasts induced by extracts of Gram-negative bacteria. J. Lab. clin. Med. 85, 422-435. Buckingham R. B., Castor C. W. and Hoag P. F. (1972) The effect of bacterial products on synovial fibroblast function: Hypermetabolic changes induced by endotoxin. J. clin. Invest. 51, 1186-l 194. Castor C. W., Bignall M. C., Hossler P. A. and Roberts D. J. (1981) Connective tissue activation-XXl. Regulation of glycosaminoglycan metabolism by lymphocyte (CTAP-I) and platelet (CTAP-III) growth factors. In Vitro 17, 777-785. Castor C. W., Fremuth T. D., Furley A. M. and Jourdian G. W. (1983) Hvaluronic acid and nroteoalvcan synthesis by lung fib&blasts in basal and aciivatedsiates. in Vitro 19, 462-470. Comper W. D. and Laurent T. C. (1978) Physiological function of connective tissue polysaccharides. Physiol. Rev. 58, 255-315. Daly C. G., Seymour G. J. and Kaiser J. B. (1980) Bacterial endotoxin: a role in chronic inflammatory periodontal disease? J. oral Path. 9, l-15. Dayer J.-M., Goldring S. R., Robinson D. R. and Krane S. M. (1979) Effects of human mononuclear cell factor on cultured rheumatoid synovial cells. Biochim. biophys. Acta 586, 87-105. Forrester J. V. and Balazs E. A. (1980) Inhibition of phagocytosis by high molecular weight hyaluronate. Immunology 40, 43545. Fratantoni J. C., Hall C. W. and Neufeld E. F. (1968) The defect in the Hurler’s and Hunter’s syndromes: faulty degradation of mucopolysaccharides. Proc. natn. Acad. Sci. U.S.A. 60, 699-706. Goldberg R. L. and Toole B. P. (1987) Hyaluronate inhibition of cell proliferation. Arthritis Rheum. 30. 769-778. Hanazawa S., Nakada K., Ohmori Y., Miyoshi T., Amano S. and Kitano S. (1985) Functional role of interleukin-1 in periodontal disease: induction of interleukin-1 production by Bacteroides gingivalis lipopolysaccharide in neritoneal macronhaaes from C3H/HeN and C3H/HeJ mice. Infect. Immk-9, 62-270. Hart G. W. and Lennarz W. J. (1978) Effects of Tunicamycin on the biosynthesis of glycosaminoglycans by embryonic chick cornea. J. biol. Chem. 253, 5795-5801. Hausman E.. Raisz L. G. and Miller W. J. (1970) Endotoxin stimulation of bone resorption in tissue culture. Science 168,862-864.
LPS and fibroblast hyaluronate Hopwood J. J. and Dorfman A. (1977) Glycosaminoglycans synthesized by cultured human skin fibroblasts after transformation with Simian virus 40. J. biof. Chem. 252, 4111-4185. Huey G., Stair S. and Stem R. (1990) Hyaluronic acid determinations: optimizing assay parameters. Matrix 10, 61-74. Kleine T. 0. (1981) Biosynthesis of proteoglycans: an approach to locate it in different membrane systems. ht. Rev. Connect. Tiss. Rev. 9, 21-98.
Kotani S. and Takada H. (1990) Structural requirements of lipid A for endotoxicity and other biological activitiesan overview. Adv. expl Biol. Med. 256, 1343. Larjava H. (1984) Metabolic change in cultured gingival fibroblasts exposed to bacterial extracts. Stimulation of hyaluronic acid synthesis. J. periodont. Res. 19, 230-237.
Larjava H. and Jalkanen M. (1984) Cell surface glycoconjugates of gingival fibroblasts exposed to dental plaque extract. J. periodont. Res. 19, 469-482. Larjava H., Jalkanen M., Pentinen R. and Paunio K. (1983) Enhanced synthesis of hyaluronic acid by human gingival fibroblasts exposed to human dental bacterial plaque. J. periodont. Res. 18, 31-39. Larjava H., Uitto V.-J., Eerola E. and Haaspasalo M. (1987) Inhibition of gingival fibroblast growth by Bacieroides gingivalis. In,&ci Immun. 55, 210-205. Manleson J. L. and Buchwald M. (1981) Effect of cvclohex&de and dexametha.sone phosphate’ on hyaluronic acid synthesis and secretion in cultured human skin fibroblasts. J. Cell Physiol. 109, 215-222. Mian N. (1986) Analysis of cell-growth-phase-related variations in hyaluronate synthetase activity of isolated plasma-membrane fractions of cultured human skin fibroblasts. Biochem. J. 231, 333-342. Millar S. J., Goldstein E. G., Levine M. J. and Hausman E. (1986) Modulation of bone metabolism by two chemically distinct lipopolysaccharide fractions from Bacteroides gingivalis. Infect. Immun. 51, 302-306. Nakamura T., Takagaki K., Kubo K., Morikawa A., Tamura S. and Endo M. (1990) Extracellular depolymer-
AOB WI I-S
191
ixation of hyaluronic acid in cultured human skin fibroblasts. Biochem. biophys. Res. Commun. 172, 70-76. Narayanan A. S. and Page R. C. (1983) Connective tissues of the periodontium: a summary of current work. Collagen rel. Res. 3, 33-64. Narayanan A. S., Page R. C. and Kuxan F. (1978) Collagens svnthesized in vitro bv dinloid fibroblasts obtained from chronically inflamed human connective tissue. Lab. Invest. 39, 61-65. Page R. C. and Schroeder H. E. (1976) Pathogenesis of inflammatory periodontal disease. A summary of current work. Lab. Invest. 33, 235-246. Prehm P. (1984) Hyaluronate is synthesized at plasma membranes. Biochem. J. 2M, 597-600. Smith G. L. (1976) Synergistic action of bacterial lipopolysaccharides on serum stimulated DNA synthesis in mouse embryo fibroblasts. Proc. Sot. exp. Biol. Med. 153, 187-192.
Toole B. P. (1981) Glycosaminoglycans in morphogenesis. In Cell Biology of the Extracellular Matrix (Ed. Hay E. D.), pp. 259-294. Plenum Press, New York.. Vaheri A., Ruoslahti E., Sarvas M. and Nurminen M. (1973) Mitogenic effect by lipopolysaccharide and pokeweed mitogen on density-inhibited chick embryo fibroblasts. J. exp. Med. 138, 13561364. Wahl L. M., &en C. E., Sandberg A. L. and Mergenhagen S. E. (1977) Prostaglandin regulation of macrophage collagenase production. Proc. natn. Acad. Sci. U.S.A. 14, 49554958.
Westphal 0. and Jann K. (1965) Bacterial lipopolysaccharide extraction with phenol water and further application of these procedures. Methodr Carbohydr. Chem. 5, 83-91. Yaron M., Yaron I., Wiletxki C. and Zor U. (1978) Interrelationship between stimulation of prostaglandin E and hyaluronate production by Poly (I), Poly (C) and interferon in synovial fibroblast culture. Arthritb Rheum. 21, 694698.
Yaron M., Yaron I., Smetana O., Eylan E. and Zor U. (1980) Stimulation of prostaglandin E production by bacterial endotoxins in cultured human synovial fibroblasts. Arthritis Rhewn. 23, 921-925.
