In vitro senescence enhances IL-6 production in human gingival fibroblasts induced by lipopolysaccharide from Campylobacter rectus

mechaniims of aging and development Mechanisms

ot‘ Ageing

and Development

87 (1996) 37 -59

In vitro senescence enhances IL-6 production in human gingival fibroblasts induced by lipopolysaccharide from Campylobacter rectus Naomi Oguraa, Utako Matsuda”, Fumimaru Tanaka”, Yasuko Shibata’, Hisashi Takiguchib, Yoshimitsu Abikob.* “L)epurtment of Oral Surger_v, Nihon tinicrrsit~

School of’ Dentistry ut Mutsdo. 2-870-1, Sakaecho-Nishi. Mutsudo. Chiha 271. Japan bDepartmerzt qf Biochrn~istry. Nihon Uniurrsit~vSrhool oj’ Dentistry at Matsudo, 2-S70- I. Sakaecho-Nishi, Received

2 October

Mutsudo. ClGba 271. Japun

1995; revised 4 January

1996; accepted

18 January

1996

Abstract

The production of interleukin-6 (IL-6) in human gingival fibroblasts (Gin cells) is increased by lipopolysacchdride (LPS) from Cumpylobacter rrctzts (C. rectus). which is associated with adult periodontitis; however, the age-related changes in the susceptibility of Gin cells to C. rectus LPS remain unclear. We examined the influence of in vitro senescence on C. rectus LPS-stimulated IL-6 production in Gin cells. LPS was prepared from C. rectus ATCC 33238 using hot phenol-water. The Gin cells were established from healthy gingival tissue removed from three patients, aged 10-12 years. The cells were cultured until confluence then stimulated with LPS (0.01. 0.1, 1.0 and 10.0 pgglml). Levels of IL-6 released in the medium were measured after incubation for 3, 6, 9, 12, and 24 h. In both young (5-6 population doublings) and senescent (17-20 population doublings) cells. LPS stimulated IL-6 production in a dose- and time-dependent manner. In response to 0.01~10.0 pg/ml of LPS. IL-6 production in the senescent cells was higher than that in the young cells. Using cells from each of the three donors, we found that this phenomenon of higher LPS-stimulated IL-6 production in senescent cell5 was reproducible. The greater capacity of the senescent cells to synthesize IL-6 in response to LPS was a higher production of mRNA for IL-6. This increase of IL-6

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48

N. guru et al. ; Mechanisms

of’ Ageing

andDecelopmenr 87 (1996)47-59

production induced by C. rectus LPS in senescent Gin cells could help to explain increased susceptibility to periodontal diseases shown by aged individuals.

Keywords: In vitro senescence; Campylobacterrectus

Gingival

fibroblasts;

Interleukin-6;

the

Lipopolysaccharide:

1. Introduction

It is widely accepted that the main causative factor of tooth loss in the aged is periodontal diseases associated with bacterial infection, the characteristic sign of these diseases being alveolar bone loss. Cltmnpylobitcter rectus (C. rectus), a Gram-negative, strictly anaerobic bacillus is associated with adult periodontitis [I]. There is, however, little information on the pathogenic potential of this bacterium. Gillespie et al. [2] have characterized C. rectus lipopolysaccharide (LPS) chemically and biologically, and have suggested that it is a strong inducer of prostaglandin E and interleukin (IL)-1 in macrophages. The release of IL-1 and other cytokines by mononuclear phagocytes is an important step in inflammatory and immune responses. These factors mobilize fibroblasts from adjacent connective tissue and stimulate local fibroblast proliferation [3]. Indeed, fibroblasts themselves have been demonstrated to produce IL-l. Sismery-Durrant and Hopps [4] have demonstrated that small amounts of IL-l,8 were released from unstimulated human gingival fibroblasts (Gin cells), and that the release of IL-lb was stimulated by LPS from Porphyromonus gingivalis (P. gingiwlis), which is also associated with adult periodontitis. Takada et al. [5] have also reported the induction of IL-lp and -6 in Gin cells stimulated with Bacteroides LPS. We have recently found that both IL-6 production and IL-6 mRNA levels in Gin cells were increased by LPS derived from bacteria associated with periodontal diseases, with the influence of LPS from C. rectus being greater than that of any other bacteria examined [6]. It is now appreciated that IL-6 is a very important polypeptide mediator that regulates, the inflammatory and immune responses. Furthermore, it has been demonstrated that IL-6 production was increased in osteoblasts treated with bone-resorbing agents such as IL-lb and LPS [7,8], and that IL-6 enhanced the formation of osteoclast-like cells 191. IL-6 may thus also be associated with bone metabolism. Human diploid fibroblasts have a limited replicative life-span in vitro, and this has been correlated with human age in vivo [lo-121. These cells have frequently been used as a model of cellular senescence, in terms of proliferative capacity [lo-121. Eleftheriou et al. [13] have suggested that the structural and quantitative characteristics of secreted proteins in human fibroblasts were modified in a similar manner during both in vivo and in vitro aging. Although C. rectus is thought to be an important pathogen in adult periodontitis, the age-related changes of Gin cell susceptibility to C. rectus LPS remain unclear. We investigated the influence of in vitro senescence on C. rectus LPS-stimulated IL-6 production in Gin cells.

N. Ogura

2. Materials

et al. /))Mechanisms

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49

and methods

2.1. Cell cultllre Gin cells were prepared according to the method of Somerman et al. [14]. Specimens of normal human gingival tissue were obtained from three patients (10, 11, and 12 years old) during premolar extraction for orthodontic treatment. The informed consent of the patients was obtained before we began the study. Each explant was placed in a 35-mm tissue culture dish and covered with a sterilized glass coverslip. The culture was performed in a-MEM (GIBCO, Grand Island, NY) supplemented with 100 unit/ml penicillin G (Banyu Pharmaceutical Co. Ltd., Tokyo, Japan), 50 Lig/ml gentamycin sulfate (Sigma Chemical Co., St., Louis, MO). 250 ng/ml amphotericin B (Flow Laboratories, McLean, VA), 5 mM HEPES buffer (pH 7.2), and 10% fetal bovine serum (FBS) under 5% CO, in air at 37°C. When the cell growth from the explant had reached confluence, the cells were detached with 0.05% trypsin (580 BAEE U/mg, GIBCO) in PBS and subcultured in a culture flask. To avoid contamination by epithelial cells, which are less easily detached than fibroblasts, some cells still attached to the bottom of the flask were discarded. Cells observed at confluence by phase-contrast microscopy had not formed the small mats typical of epithelial cells. Young Gin cells were defined as those with 5-6 population doublings and senescent Gin cells as those with 17-20 population doublings. Their current expected life-span is approximately 30 population doublings. 2.2. Bucterid

culture and LPS preparation

C. rectus (ATCC 33238) was cultured in Todd-Hewitt broth supplemented with 0.5% yeast extract, 0.2% ammonium formate, and 0.3% sodium formate at pH 7.6, as described by Kokeguchi et al. [15], in an anaerobic chamber containing SO’/0 N1, 10% Hz, and 10% CO,. LPS was extracted and partially purified from bacteria by the method of Koga et al. [16]. Briefly, lyophilized cells (8 g) were suspended in 280 ml of pyrogen-free water and 280 ml of 90% phenol. The mixture was stirred vigorously at 65°C for 20 min and then centrifuged at 7000 x g for 20 min. The aqueous phase was removed, and the phenol phase and insoluble precipitate were re-extracted with 280 ml of pyrogen-free water. The combined solution was dialyzed extensively against distilled water and lyophilized; this we termed crude phenol-water-extracted LPS. The crude LPS (1 g) was suspended in 100 ml of pyrogen-free water and centrifuged at 100000 x g for 3 h. The pellets were suspended in 20 ml of 10 mM Tris-HCl buffer (pH 7.4), containing 0.1 mM ZnCl, and 400 /lg of nuclease Pl from Prniciflunz citrinum (Yamasa Shoyu Co. Ltd., Chiba, Japan). The reaction mixture was incubated at 37°C for 16 h and then dialyzed extensively against distilled water. The dialyzed solution was centrifuged at 100 000 x g for 3 h. and the pellets were lyophilized.

2.1. Itntnunoassu~~ oj’ IL -6 Young and senescent Gin cells were plated, at 1 x 1Oi cells (0.5 ml medium) per well, in 24-well plates. Confluent stage cells were then incubated for 24 h in medium containing 2% FBS, after which, the cells were treated with C. wws LPS (1 fig/ml) at 37°C for 12 h. The concentration of IL-6 in the conditioned medium was determined with a two-step sandwich immunoassay performed with a human IL-6 ELISA reagent kit (HYCYTE, Uden, Netherlands). A 96well plate was coated with anti-human IL-6 monoclonal antibody at 4°C for 18 h. Blocking with 5% skim milk was performed and the samples were incubated for 2 h. The plate was washed four times, anti-human IL-6 polyclonal antibody was added, and the incubation was continued for a further 1 h. The next step consisted of l-h incubation with horseradish peroxidase-conjugated second antibody. Peroxidase activity was developed with TMB reagent (HYCYTE) and the optical density of each well was measured at a wavelength of 415 nm. Basal IL-6 production was defined as IL-6 production in the absence of LPS and total IL-6 productions was defined as IL-6 production in the presence of LPS in the culture medium. The difference between total IL-6 production and basal IL-6 production was regarded as LPS-stimulated IL-6 production. 1.4. RT-PCR

analysis

Acid-guanidinium thiocyanate-phenol-chloroform extraction was used to extract total cellular RNA from young and senescent Gin cells [17]. Guanidinium thiocyanate (4 M) containing 0.1 M 2-mercaptoethanol, was added to untreated Gin cells and to Gin cells treated with LPS, and the mixture was homogenized. Sodium acetate (2 M), phenol, and chloroform were then used to extract the RNA, which was then precipitated with isopropanol at - 20°C for 1 h, after which the precipitate was redissolved and reprecipitated in isopropanol at - 20°C for 1 h. The final RNA precipitate was stored in ethanol at - 135°C. cDNA synthesis and amplification by reverse transcription-polymerase chain reaction (RT-PCR) were carried out with a GeneAmp RNA PCR kit (PerkinElmer, New Jersey, USA). Briefly, cDNA synthesis was carried out at 42°C for 15 min in a final volume of 20 jtl contained 4 ~11 of MgCl? Solution (25 mM), 2 ~11of 10 x PCR Buffer II (500 mM KCl, 100 mM Tris-HCl, pH 8.3). 2 ~11of dNTP (10 mM each), 1 /ll of RNase inhibitor (20 U//11), 1 ~1 of MuLV reverse transcriptase (25 U/pi), 1 ~11 of random hexamers (25 /IM), 1 ~11 of oligo d(T),6 (25 LIM), and 2 ~1 of total RNA (1 pg//ll). The PCR mixture, containing 20 ~1 of the cDNA solution, 4 ~1 of 25 mM MgCl,, 8 /il of 10 x Buffer II, 1 ~1 of forward primer, 1 it1 of reverse primer, 65.5 ~11 of H,O, and 0.5 ~1 of AmpliTq DNA polymerase, was subjected to amplification with a GeneAmp PCR system 9600 (Perkin-Elmer) set at 94°C for 1 min, 55°C for 2 min, and 72°C for 2 min, for 25535 cycles. The primers for IL-6 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were synthesized. The designed primers were: 5’-GCG CAG AAT GAG ATG AGT-3’ ( 169-186. forward primer for IL-6);

h’. Ogura et al. ///Mechanisms of Ageirtg and Deoelopment 87 (1996) 47-59

51

5’-CCA CTC ACC TCT TCA GAA-3’ (6044621, reverse primer for IL-6); 5’-ATC ACC ATC TTC CAG GAG-3’ (forward primer for GAPDH); and 5’-ATG GAC TGT GGT CAT GAG-3’ (reverse primer for GAPDH). PCR fragments were electrophoresed on 1.5% agarose gel, and subsequently stained with ethidium bromide.

3. Results 3. I. Time course of IL-6 production We examined the effect of incubation time on the production of IL-6 in young and senescent Gin cells in the absence of C. rectus LPS. Regardless of the length of incubation time, the constitutive production of IL-6 in the absence of LPS (basal IL-6 production) in both the young and senescent cells was very low (Fig. 1A) and the basal IL-6 production was not markedly increased in the senescent cells relative A (without LPS)

E-

0

1o

6

12

18

24

12

1X

24

c (B-A)

I

0

6

Time (h) Fig. 1. Time course of IL-6 production in young and senescent Gin cells (cell I) in the absence and presence of C. rectus LPS (I fig/ml). Young cells (0): senescent cells ( -8). Each point is the mean of triplicate cultures. (A) Basal IL-6 production, (B) total IL-6 production. and (C) LPS-stimulated IL-6 production.

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IV. Ogura et al. / Mechanisms of Ageing and Development 87 (1996) 47- 59

LPS concentration(@ml) Fig. 2. Dose response curve of IL-6 production in senescent cells with increasing amounts of LPS from C. YL’CZUS LPS. Basal IL-6 production (U): total IL-6 production (0); and LPS-stimulated IL-6 production (0). Each point is the mean of triplicate cultures. The cultures with the indicated concentrations of LPS were incubated at 37°C for 12 h.

to the young cells. In the presence of LPS, total IL-6 production in both the young and senescent cells increased with incubation time after 12 h (Fig. 1B). The LPS-stimulated IL-6 production in both young and senescent cells occurred in a time-dependent manner after 12-h incubation (Fig. 1C). LPS-stimulated IL-6 production in the senescent cells for each incubation period observed was higher than that in the young cells. 3.2. EfJ;t of LPS concentration on IL-6production The effects of LPS concentration on LPS-stimulated IL-6 production in the senescent cells are shown in Fig. 2. IL-6 production uniformly increased from 0.01 pug/ml LPS to maximum stimulation at 1.0 pg/ml: at 10.0 pg/ml LPS, LPS-stimulated IL-6 production shcwed no further change remaining at the maximal level. LPS-stimulated IL-6 production in the young cells was dose-dependent between 0.01 and 1.0 /fg/ml, and LPS-stimulated IL-6 production in the presence of various concentrations of LPS (0.01, 0.1, 1.0 and 10.0 pg/ml) was higher in the senescent than in the young cells (data not shown). From the results shown in Figs. 1 and 2, we selected the optimum conditions for the remainder of our experiments. Unless otherwise specified, we performed 12-h incubation in the absence and presence of 1.0 pg/ml LPS to assess LPS-stimulated IL-6 production in both young and senescent cells. 3.3. Reproducibility of LPS-stimulated IL-6 production

As the results shown in Figs. 1 and 2 suggested that LPS-stimulated IL-6 production in the senescent cells was greater than that in the young cells, we examined the reproducibility of the enhanced LPS-stimulated IL-6 production in

A’. Ogura et al.

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the senescent cells. Both young and senescent cells were prepared from three healthy young donors. We found that the basal, total and LPS-stimulated IL-6 production in the senescent cells from all three donors was higher than the level in the young cells (Fig. 3). Of note, LPS-stimulated IL-6 production in the senescent cells was significantly higher (2.4 to 5.4-fold) than that in the young cells (Fig. 3C) and basal IL-6 production in the senescent cells was slightly increased (1.2 to 2.1-fold) compared to that in the young cells (Fig. 3A). When viewed from the standpoint of the comparison of the amounts of the basal and LPS-stimulated IL-6 production, although there is a negligible difference between each the young cells, the amount of LPS-stimulated IL-6 production in the senescent cells was 2.6 to 3.1-fold that in young cells (Fig. 3A and 3C).

Cell

1

2

3

1

2

2

3

1

2

.

3

10 IC (B-A)

Cell

1

3

Fig. 3. Effect of in vitro senescence on C. reczus LPS-stimulated IL-6 production in Gin cells. The cells were prepared from three healthy individuals and were incubated wsith or without C. YC’L’~US LPS (1.0 pcg:ml) at 37°C for 12 h. Results in A and B are means i SE. (n = 4). *p< 0.05, **p < 0.01, compared to basal IL-6 production. ***p < 0.001. compared to total IL-6 production by the young cells from each donor. Results in C are means (?I = 4). (A) Basal IL-6 production, (B) total IL-6 production, and (C) LPS-stimulated IL-6 production.

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GAF’DH Fig. 4. ElTect of in vitro senescence on C. rectu.s LPS-stimulated IL-6 mRNA expression in Gin cells (Cell 1). The cells were incubated with or without LPS (1.0 pcgiml) at 37°C for 12 h, after which RT-PCR analysis was carried out. I, untreated young cells; _, 7 young cells treated with LPS; 3, untreated senescent cells: 4. senescent cells treated with LPS.

3.4. Relutive

expression

of mRNA

for IL-6

Examination of the relative expression of mRNA for IL-6 in the young and senescent cells by RT-PCR analysis showed low levels of IL-6 mRNA expression in the untreated young cells and increased expression in the untreated senescent cells compared with the young untreated cells (Fig. 4). C. rectus LPS increased the relative level of mRNA for IL-6 in both the young and senescent cells compared to untreated cells, however, the level of IL-6 mRNA in the senescent cells was higher than that in the young cells (Fig. 4).

4. Discussion Since the number of cell divisions before the onset of senescence is inversely correlated with the age of the tissue donor [ 10,111, we prepared fibroblasts from the gingiva of three young patients lo- to 12-years-old. Hayflick and Moorhead [lo] have clearly shown that normal cultured human cells have a finite capacity for replication in vitro. We determined the life-span of the Gin cells used in this study in advance, and found that the cells underwent about 30 population doublings before division ceased. Accordingly, we defined as young cells those with 5-6 population doublings and senescent cells as those with 17-20 population doublings. Garrison and Nichols [18] have reported that LPS preparations obtained from Scrlmonellrr typhinzuriurn, Bucteroides intermedius and P. gingivulis, using hot phenol-water, contained detectable levels of protein, measured by the Lowry method, but that protein was undetectable in C. rectus and Actinobacillus nctinomycetemcamitans (A. actinomycetemcomitans) LPS preparations. As we considered that protein contaminants associated with LPS preparation could participate in stimulating IL-6 production in Gin cells, we examined protein contamination in our LPS preparations beforehand, using the method of Bradford [19]; protein was undetectable in the C. rectus LPS preparation used in this study (data not shown). This finding is consistent with the observations of Garrison and Nichols [18] described above. It has been demonstrated that IL-6 by exerting multiple biological functions, plays a key role in immune responses, bone formation, and hematopoiesis, and that

the abnormal production of IL-6 is linked to various diseases 1201. Recent reports have shown that, in several mammalian species, including humans, IL-6 protein levels measured in the plasma of healthy elderly individuals are elevated compared to levels in young individuals [21,22]. Wei et al. [24] suggested that the increased plasma IL-6 levels may be of importance in the vulnerability to illnesses such as commonly shown in elderly people. Fagiolo et al. [23] have demonstrated significant increases of IL-6 levels in mitogen-stimulated peripheral mononuclear cells obtained from aged donors compared to levels in cells from young donors, and this finding may be relevant to several aspects of age-associated pathological events, including atherosclerosis, osteoporosis, fibrosis, and dementia. Vandenabeele and Fiers [24] proposed that amyloidogenesis in Alzheimer’s disease in the elderly may be due to an acute phase response, mediated by IL-6, which possibly induces the production of /I-amyloid protein precursors. In our previous studies, we have found that IL-6 production and IL-6 mRNA levels in Gin cells [6] and periodontal ligament cells [25] were enhanced by LPS obtained from Gram-negative bacteria associated with periodontal disease. Yamazaki et al. [26] have also reported that IL-6 production by Gin cells was stimulated by LPS, from P. gingivalis, which organism is associated with periodontal disease. Of note, significantly higher levels of IL-6 have been detected in the inflamed gingiva of patients with periodontitis compared to levels in healthy controls [27,28]. Agarwal et al. [29] have very recently reported that the production of IL-6 and the expression of its mRNA in human monocytes was increased by LPS obtained from A. actinomycetemcomitans and P. gingicalis. These findings suggest that IL-6 plays an important role in periodontal disease. Further, levels of IL-6 in the presence of periodontal diseases seem to be elevated to a greater extent in older individuals than in young individuals. Thus, it is possible that changes in IL-6 levels contribute to the increased incidence of periodontal diseases in older individuals. In this in vitro study, we presented evidence of a significant increase in C. rectus LPS-stimulated IL-6 production in senescent Gin cells compared to young cells, and this increase was time- and dose-dependent. When the Gin cells were cultured in the presence of Bacteroides LPS, the IL-6 produced was detected mainly in the supernatant of the culture medium [5]; so we therefore assayed IL-6 in the conditioned medium of the Gin cells, regarding this as IL-6 synthesized by the cells. In order to confirm the reproducibility of our finding that LPS-stimulated IL-6 production in the senescent cells was increased to a greater extent than in the young cells, we quantitatively compared LPS-stimulated IL-6 production in the young and senescent cells of three individuals, under different time and dose conditions. Although the levels of LPS-stimulated IL-6 production in both the young and senescent cells obtained from the three individuals, was varied from donor to donor, the LPS-stimulated IL-6 production in the senescent cells was significantly higher than that in the young cells for each individual. We next compared the level of IL-6 mRNA expression in the young and senescent cells 12 h after the addition of LPS (1.0 pg/mll. Fig. 4 shows that the level of IL-6 mRNA expression was higher in the senescent than in the young cells. These findings suggested that the greater capacity of the senescent cells to produce IL-6 in response to C. rectus LPS

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is due to the greater level of IL-6 mRNA synthesis. Earlier studies have indicated that LPS levels in gingival crevicular fluid are positively correlated with clinical and histologic signs of gingival inflammation [30,31]. It has been also reported that LPS can penetrate gingival tissues [32,33]. Furthermore, Sismey-Durrant and Hopps [4] have observed that a low concentration of P. gingiuulis LPS (0.1 pg/ml) produced PGE, in Gin cells. and they also suggested that this level of LPS may bring about PGEz production in Gin cells in vivo. Our findings that C. rectus LPS at a concentration of 0.1 /Lgg/mlstimulates IL-6 production and IL-6 mRNA expression in Gin cells indicate that C. rrctus LPS could stimulate IL-6 production in Gin cells in vivo in the aged. Since the importance of the interaction between IL-I p and IL-6 has been increasingly recognized recently, we discuss the contribution of IL-lp to IL-6 production. Navarros et al. [34] have suggested that IL-6 gene activation may occur through an autocrine regulation mechanism or be induced by early-responding cytokines, such as IL-la. We have recently reported that IL-6 production was stimulated by IL-lb in human periodontal ligament cells in vitro [35]. Zhong et al. [36] have speculatively divided the cytokines into two groups in terms of their gene expression: those showing early expression and more sensitivity to LPS, such as IL-lb, and those showing delayed expression and less sensitivity to LPS, such as IL-6. They have speculated that the second group of genes are activated as a result of either activation of the first group of genes or activation of their cytokine products. This idea raises the possibility that the increase in IL-6 production with LPS may occur through an increase of IL-ID production with LPS. Furthermore, Kuida et al. [37] have observed that adherent monocytes from mice harboring a disrupted gene for the IL- lp converting enzyme, which processes the inactive IL-ID precursor to the proinflammatory cytokine, did not produce IL-lb after stimulation with LPS, and the production of IL-6 in these cells remained at about 50% of that of the unstimulated control. From the findings described above, the following scenario may be considered. There are at least two mechanisms of IL-6 production: one, independent of LPS, and one, inducible by LPS. We are not of the opinion that IL-lfi is the sole cause for the stimulation of IL-6 production. However, in the light of own observation that LPS-stimulated IL-IF production was higher in senescent than in young Gin cells (unpublished data), we suggest that one of the causes for the increased LPS-stimulated IL-6 production in the senescent Gin cells may be an increase in the production of IL-IB, which cytokine is also induced by LPS. Recently, it has been reported that LPS receptors that probably bind to lipid A, are presented on cells of monocyte lineage [38]. Several studies have shown that the interaction of LPS with monocytes involves a plasma protein, LPS-binding protein (LBP), that binds to LPS, and a glycosylphosphatidylinositol-anchored cell-surface glycoprotein, CD14, that efficiently binds LPS when it is presented as an LPS-LBP complex [39,40]. Furthermore, Han et al. [41] have demonstrated that LPS induced the tyrosine phosphorylation of a 38-kDa protein in a murine pre-B cell line. This phosphorylation was induced by the R- and S-forms of LPS, as well as by synthetic lipid A. On the other hand. Geng et al. [42] reported that the signal transduction

pathways responsible for LPS induction of IL-6 gene expression in blood monocytes were related to the activation of protein tyrosine kinase (PTK) and protein kinase A (PKA). In fibroblasts, the cellular regulatory machinery has not yet been defined, although it has been shown by many investigators that fibroblasts are responsive to LPS [4,5,25,26,28]]. Lei et al. [43] reported that fibroblasts presented LPS receptors on the cell surface; however, whether or not the protein was CD14 was not clarified. It is necessary both to clarify the mechanism responsible for the signal transduction pathways of LPS and to biochemically characterize LPS receptors in fibroblasts. Further work is also required to examine LPS receptors and the mechanism responsible for the signal transduction pathways of LPS in Gin cells to elucidate the mechanism whereby IL-6 production is increased by senescence. We will be examining this problem in further studies. Destructive periodontal disease has been so consistently associated with aging that many have come to see it as an inevitable consequence of growing older. Brian and Burt [44] reported a linear relationship between age and some loss of periodontal attachment. Van der Velden [45] has suggested that there are age-related changes in the periodontium, and that, with age, the periodontium reacts differently to plaque, with inflammation developing more rapidly and healing proceeding more slowly. Papapanou and Lindhe [46] have indicated, however, that intact supporting periodontal tissue can be retained in elderly subjects, and they have corroborated findings that loss of clinical attachment and alveolar bone is not a necessary consequence of aging. The different findings in these studies may reflect individual variations in randomly selected human subjects. On the other hand, the in vitro senescence of cultured human fibroblasts is widely accepted as a model for in vivo aging [ 13,47.48]. Therefore, we conclude that in vitro senescent Gin cells are useful for studies of the relationship of periodontal diseases and aging. In conclusion, the increase of IL-6 production induced by LPS in senescent Gin cells compared to IL-6 production in young cells may help to explain the increased susceptibility to periodontal diseases shown by older people.

Acknowledgements This work was supported, in part, by Funds for Comprehensive Research on Aging and Health from the Ministry of Public Welfare of Japan (93A2311) and a Grant-in-Aid for Encouragement of Young Scientists ( # 07771984) from the Ministry of Education, Science and Culture of Japan.

References [I] J.L. Dzink, A.C.R. Tanner, A.D. Hatrajee and S.S. Socransky. Gram negative species associated with active destructive periodontal lesions. J. Clin. Periodmtol., 12 (1985) 648-659. [It] J. Gillespie, S.T. Weintraub. G.G. Wong and S.C. Holt, Chemical and Biological characterization of the lipopolysaccharide of the oral pathogen W’ohella rec’tu ATCC 33238. I&t. IMMU~I.. 56 (1988) 2028 -2035.

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