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APMIS, 1996, vol. 104, (5), pp 367-373

The interference  of gingival cell cultures  with growth of selected bacteria

JOHANSSON, ANDERS¹; BERGENHOLTZ, AXEL¹; HOLM, STIG E.²

ABSTRACT

The aim of the present study was to analyze the interference of oral tissue cells or cell lines (effector cells) with growth of reference bacteria, and furthermore to investigate whether cells derived from different individuals differ in such activity. The reference bacteria were Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mitis, Actinobacillus actinomycetemcomitans, Porphyromonas gingi-valis, and Fusobacterium nucleatum. The effector cells used were gingival fibroblasts (GF) from 21 periodontally involved persons, gingival epithelial cells (E) from 2 such persons, HeLa cells (HeLa), and an amnion cell line (Amnion). The cells were cultivated and their supernatants tested for antibacterial activity in a Bioscreen robot analyzer (Labsystems, Finland). Results suggest that the antibacterial activity of each tested primary cell line of tissue had its own profile depending on cell type and donor, and that the composition of oral microbiota was influenced by oral cells, which might, in turn, contribute to the variations in the pathogenesis of periodontal diseases.

Key words: Gingival fibroblasts; bacterial interaction; gingival epithelial cells; Bioscreen.

INTRODUCTION

Specific as well as nonspecific defense mechanisms act to protect the integrity of the oral surfaces (for review see 23). Normally there is harmony between the existing oral microbiota and the host with the balance of microbial interactions resulting in microbial homeostasis and microbial stability. When imbalances occur, the defense systems serve to maintain the microbial homeostasis, preventing colonization and invasion of the oral tissue by pathogens. The nonspecific defense factors such as salivary flow, mucin and agglutinins physically remove the bacteria (1, 10), while others such as the lysozyme-protease-monovalent anion antibacterial system and human iron-free (apo) lactoferrin (through their bactericidal activity) cause cellular lysis and killing (2, 3, 20). Histidine-rich peptides are active against bacteria and fungi (26, 27). Specific defense factors including intraepithelial lymphocytes and Langerhans cells, IgG and IgA form a barrier restricting bacterial and/or antigenic penetration (23).

Disturbances in leukocyte functions associated with periodontal diseases have been shown to be normalized by successful treatment (7, 11).

Recently, there have been reports of the existence of antimicrobial peptides in mammalian mucosal surfaces. While in 1992 Jones & Bevins (19) isolated an antimicrobial peptide gene expressed by human epithelial cells, no clear pattern has emerged as to which mammalian mucosal surfaces are so protected, and whether such peptides are produced by epithelial cells or fibroblasts. An epithelial antimicrobial peptide isolated from bovine tongue was reported in 1995 by Schonwetter et al. (29).

The suggested role of mucosal defense proteins is to protect the body from disease by regulating the level and composition of the microflora, and restricting microbial invasion of the mucosa (5, 18). Such mechanisms may be of significance in determining the specific tropism of bacteria for various host surfaces in the oral cavity.

Additionally, the immunoglobulins (IgG, IgA, IgM) act as opsonins, activating complement and preventing bacterial adhesion (23, 34). It has also been shown that specific and non-specific defense factors may function synergistically (22).

The aims of the present study were to confirm the interference of human gingival cells with growth of various bacteria, and to investigate how such interference varies from individual to individual.

Staphylococcus aureus (Sa) ATCC 6538, Staphylococcus epidermidis (Se) ATCC 12228, Streptococcus mitis (Sm) clinical isolate a5 (13), Actinobacillus actinomycetemcomitans (Aa) ATCC 43718, Porphyromonas gingivalis (Pg) ATCC 33277, and Fusobacterium nucleatum (Fn) clinical isolate BN11a-d (6).

Gingival fibroblasts (GF), primary tissue from 21 periodontally affected patients; gingival epithelial cells (E), primary tissue from 2 periodontally affected patients; HeLa cells (HeLa), cervical carcinoma CCL2 (Flow Laboratories, UK), and Amnion cells (Amnion), human amnion CCL21 (Flow Laboratories, UK).

Each of the bacterial strains was cultivated on blood agar plates, except for P. gingivalis, which was cultivated on blood agar plates supplemented with hemin and vitamin K (BGA plates) (15).

For liquid cultivation, Eagle's minimal essential medium (E-MEM) solution (Flow Laboratories, UK) without phenol red and antibiotics, with 25% tryptose yeast (TY) medium 14, 10% fetal calf sera (Flow Laboratories, UK), and 20 mM HEPES (Sigma, USA), was used for the bacterial strains and cells. This culture medium was designated CM.

Quantification of bacteria in suspension was done by reading the optical density (OD) at 500 nm on a spectrophotometer (Vitatron CMP, Vital Scientific, Netherlands).

The bacterial strains were cultivated aerobically (Sa, Se, Sm) or anaerobically (Aa, Fn, Pg) on agar plates at 37°C for 24-48 h. Inoculates were then transferred to 4 ml CM in 10 ml glass tubes and incubated for 20-24 h at 37°C to the stationary phase. From the bacterial suspensions, 20 µl for Sa, Se or Sm and 160 µl for Aa, Fn or Pg was transferred to new tubes containing 4 ml CM and incubated for 4-8 h at 37°C to log phase (OD500 nm 0.5-1.0).

Gingival explants were obtained following surgery from 21 patients with a history of periodontal disease. The gingival tissue was minced and transferred to 25 cm² tissue culture flasks (Falcon, USA) on plasma clots (10 pieces in each flask). After 30 min, 5 ml E-MEM (Flow Laboratories, UK) supplemented with 100 µg penicillin-streptomycin (PEST) (Sigma, USA), 50 µl L-glutamine (Flow Laboratories, UK), and 10% FCS was added and the tissue left to grow.

Biopsies from gingiva were collected and handled in the same way as described for the gingival fibro-blasts. The epithelial cells were separated from the fibroblasts by using a selective growth medium, namely keratinocyte growth medium (KGM, Promo-cell, Germany).

Gingival fibroblasts and the cell lines (HeLa and amnion) were cultivated in E-MEM supplemented with PEST, L-glutamine, and 10% FCS, at 37°C in 5% CO². Gingival epithelial cells were cultivated in complete KGM. Growth medium was changed every 48 h. Confluent cell layers were treated for 10 s with 0.2% EDTA (KEBO-Lab, Sweden) in PBS buffer, and for 1 min with 0.1% trypsin (Flow Laboratories, UK) in PBS buffer. The contents of each flask were suspended in fresh growth medium and transferred to three new flasks. Primary cell cultures used as effector cells were in the third to eighth passage.

For antimicrobial tests, supernatants were taken from cultured cells incubated for 24 h in 35 mm cell culture petri dishes, with 4 ml CM/dish. The cell concentrations used during incubation were 4X105/ml CM for the carcinoma cell lines (HeLa and Amnion), 1 x 10⁵/ml CM for the fibroblast lines, and 2x 10⁵/ml CM for the epithelial cell lines.

A Bioscreen robot analyzer (Labsystems, Finland) was used for monitoring microbial growth in microtiter plates (Honeycombplates, Labsystems, Finland). The bacterial growth in each well was presented as turbidometric curves (growth curves). Changes in optical density of the various bacterial cultures were registered every 10 min for 24 h.

In the present study the growth of strains of six bacterial species, four of which are commonly found in the oral cavity, was analyzed. The interaction with the various oral tissue cells and cell lines (stimulated or not) was determined.

Three hundred µl of supernatant from the confluently growing mammalian cells (24 h cultures) was added to each of the wells in the Bioscreen microtiter plates, after which 100 µl of viable log phase bacteria (Sa, Se, Sm 10⁶/well, and Aa, Pg, Fn 10⁷/well) in CM was added to each well. The bacterial suspensions were incubated for 24 h at 37°C in the Bioscreen analyzer and the growth recorded for 24 h. For estimation of bacterial growth by the Bioscreen analyzer under anaerobic conditions, the microtiter plates were sealed with vacuum grease (Fluka, LabKemi, Sweden) before being transferred to the Bioscreen incubator.

The data recorded in the Bioscreen were treated as described by Johansson et al. (16).

The parameter growth density (GD), based on the kinetic bacterial growth curves, was used to show interactions between the mammalian cells and the various bacterial species. This parameter reflects the interference when the logarithmic growth of the untreated bacteria of each species was reduced by 15% of its maximum growth (Fig. 1). Interference data were presented in index form. The mean value of bacterial growth registered in control cuvettes was set at index 100 for the respective bacterial strains, and samples with cell culture medium added were always compared with the control index.

Fig. 1. Growth curve of S. epidermidis without interfering cell culture supernatants added (to exemplify the parameter Growth Density). Relative growth - kinetic doubling rate of the growing bacteria. Absolute growth - linear increase in OD at 450 nm of the growing bacteria.
 

In the figures presented, the mean values and standard errors (SE) of the six experiments were used for each growth index value.

Significant influence of various cell culture supernatants on bacterial growth was estimated by unpaired t test (p value <0.05 indicated by *).

The interference of growth medium supernatants collected from different cell types from various individuals was estimated. Their effect on each of the six tested reference bacteria is shown in Figs. 2 and 3.

Fig. 2. Interference by supernants of 24 h incubated cell cultures from different cell types from various individuals with growth of oral reference bacteria. Mean±SE n=3, unpaired t test p value <0.05 indicated by *. Cultured cells: GF=gingival fibroblasts, E=gingival epithelial cells, HeLa=HeLa-cells, Amnion=amnion cells. Oral reference bacteria: Aa=A. actinomycetemcomitans, Fn=F. nucleatum,Pg=P. gingivalis, Sm=S. mitis.
 

Fig. 3. Interference by supernatants of 24 h incubated cell cultures from different cell types from various individuals with growth of skin reference bacteria. Mean±SE n=3, unpaired t test p value <0.05 indicated by *. Cultured cells: GF=gingival fibroblasts, E=gingival epithelial cells, HeLa=HeLa-cells, Amnion=amnion cells. Reference bacteria: Sa=S. aureus, Se=S. epidermidis.
 

A. actinomycetemcomitans (Aa) was significantly inhibited by 1 of the 21 tested fibroblast lines and by 1 of the 2 tested epithelial cell lines. One of the fibroblast lines significantly stimulated the growth of Aa (Fig. 2).

F. nucleatum (Fn) was significantly inhibited by 4 of the 21 tested fibroblast lines and by the amnion cells (Fig. 2).

P. gingivalis (Pg) was significantly inhibited by 6 of the 21 tested fibroblast lines, and by 1 of the 2 tested epithelial cell lines, and by the amnion-and HeLa cell lines. One of the fibroblast lines significantly stimulated the growth of Pg (Fig. 2).

S. mitis (Sm) was significantly inhibited by supernatants from all the tested cell cultures, except two of the fibroblast lines Fig. 2.

The growth of S. aureus (Sa) and S. epedermidis (Se) was almost completely inhibited by media from the two tested epithelial cell lines, while media from the fibroblast lines did not show a significant stimulatory effect, except in the case of one fibroblast line that significantly inhibited Se Fig. 3.

The human mouth shows various types of mucosal surfaces with remarkable tropism for bacterial colonization (12). The composition of the periodontal microflora is complex (25) with great individual variability (23, 24, 28). Periodontal diseases have been claimed to be multi-factorial in nature (4), but the composition of the periodontal microflora seems to be significant for the progression of periodontal diseases (21, 25, 31). The bacterial colonization of the mouth as well as the periodontal crevice depends on the local environment, which might be of importance in the regulation of its microflora and virulence of the plaque (31). It might be assumed that the situation in the mouth is similar to that reported for mucosal tissue cells in the human bowel and air passages containing antimicrobial peptides participating in the regulation of the microflora (9, 18, 19). However, such activity is still unknown in the human oral cavity, while from bovine tongue an epithelial antimicrobial peptide has been isolated (29).

In the present study, interaction between various mammalian cells and some bacterial species was estimated with a Bioscreen robot analyzer, registering bacterial growth cultivated in supernatants taken from the various cell cultures. Twenty-one gingival fibroblasts cultures and two gingival epithelial cell lines were tested. Two established cell lines (HeLa and amnion cells) were used as references. The six different bacterial species tested in the present study (A. actinomycetemcomitans, P. gingivalis, F. nucleatum, S. aureus, S. epidermidis and S. mitis) were selected based on their differences in virulence, growth demands and peridontopathic aspects.

Data from the present study show that gingival fibroblasts release factors in the growth medium that significantly reduce growth of some bacterial species. Each tested cell line from primary tissues had its own profile of antibacterial activity which was related to cell type and donor. The level of the various significant antibacterial effects is comparable with that seen when a [beta]-hemolytic streptococcus strain is inhibited by an [alpha]-hemolytic strain (13, 16).

The supernatants from the two tested gingival epithelial cell lines almost completely inhibited the growth of S. aureus and S. epidermidis, while supernatants from the various fibroblasts showed a stimulatory effect. This phenomenon might explain why these two bacterial species are common in the normal human skin micro-flora, but rare in the oral cavity (8). The stimulatory or weak inhibitory effect produced by supernatants from fibroblasts may, on the other hand, indicate a risk for staphylococcal infections when the oral mucosa is damaged or diseased.

The present results indicate an interaction between gingival tissue cells (fibroblasts and epithelial cells) and bacteria that may be part of the regulatory mechanisms of the oral micro-flora as well as of the colonization of the oral mucosa and the periodontal crevices. However, the question why there is a variation in the ability of gingival cells to inhibit growth of different bacterial species is still not answered. Genetic factors (24, 28) or variation in the local environment (31) are two other contributory factors which need to be further analyzed.

Ongoing studies will clarify whether the above-mentioned phenomenon can be triggered by the presence of certain microorganisms and whether the antimicrobial effects can be attributed to specific molecules.

The nature and severity of periodontal diseases may thus not be defined simply as the presence of periodontal pathogens, but most likely as a function of interactions between common bacteria and the host 25, 31. The prevalence and composition of periodontal plaque is a factor of significance for disease progression 21, 30, 32. Results from the present study indicate that the gingival tissue cells contribute to the regulation of the oral microflora. Recent data 9, 18, 19, 29 have shown the presence of antimicrobial peptides in the mucosal surfaces of the human bowel and bovine air passages. This supports the hypothesis that antimicrobial peptides contribute to the host defense.

Mucosal defense proteins may influence the level and composition of the microflora and protect the mucosa from microbial invasion 5. Detailed mechanisms are still unknown in the human oral cavity and in the periodontal crevices, but such phenomena might be of importance in elucidating the specific tropism of bacteria colonizing various host surfaces in the human mouth 12. Further investigations need to be performed.

We thank Dr. Ridwan Omar, College of Dentistry, King Saud University, Riyadh, Saudi Arabia, for correcting the English in the present manuscript.

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