a Infectious Diseases Research Group, Departments of Microbiology and Immunology and Internal Medicine, Rega Institute for Medical Research, K. U. Leuven, Minderbroedersstraat 10, 3000 Leuven; b ENT Department, K. U. Leuven, Leuven, Belgium
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Abstract |
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Introduction |
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Apart from the antimicrobial action of the drug, direct interactions between the antibiotic and host tissues may be important since they may affect the outcome of treatment. The interference of macrolides with the natural effectors involved in host defences and inflammation is an area of current interest.1 It has been shown that erythromycin inhibits respiratory glycoconjugate secretion from human airways and improves the movement of cilia on airway epithelium in vitro.2,3 It has been suggested that the inhibition of interleukin-8 (IL-8) by long-term, low-dose macrolide therapy is one of the key interactions by which chronic airway inflammation is resolved.4 The adhesion of Pseudomonas aeruginosa and Moraxella catarrhalis can be inhibited by erythromycin but it is not known whether this results from interaction between erythromycin and the epithelial cells or the bacteria.5
The aim of our study was to determine whether subinhibitory concentrations of erythromycin affected pneumococcal adhesion to respiratory epithelium, damaged respiratory epithelium or affected secretion of IL-8 by the epithelial cells. Therefore, we developed an in vitro cell model expressing characteristics of a differentiated respiratory epithelium in which these effects can be studied.
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Materials and methods |
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The S. pneumoniae isolate used was obtained from a blood culture and belonged to serotype 23. The MIC of erythromycin, as assessed by Etest (AB Biodisk, Solna, Sweden), was >256 mg/L. PCR confirmed the presence of the ermAM gene.6 The isolate was also resistant to penicillin (MIC = 3 mg/L).
Cell isolation and culture conditions
Nasal epithelial cells were isolated by enzymic dissociation of tissue from nasal polyps and paranasal sinus mucosa. Tissue was obtained during surgery on patients suffering from polyposis nasi or chronic sinusitis. The cells were cultured by a modification of the techniques described by Jorissen et al.7 and Yamaya et al.8 The culture medium consisted of a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 with high glucose (4500 mg/L) (Gibco BRL, Life Technologies, Rockville, MD, USA) supplemented with 2% Ultroser G (Gibco), cholera toxin 10 µg/L (Sigma-Aldrich, Bornem, Belgium), penicillin (50 IU/mL) and streptomycin (50 mg/L). The tissue fragments were washed three times in saline, then exposed to 0.1% Pronase (Sigma-Aldrich) in culture medium (without Ultroser G) for 1624 h at 4°C under continuous rotation. After a single wash with Nu-serum (Becton Dickinson, Bedford, MA, USA) to inactivate the Pronase and two washes with culture medium, the cell suspension was preplated for 1 h at 37°C on to plastic (Tissue Flask T25, Corning Costar, Acton, MA, USA) to reduce the number of contaminating fibroblasts. The cell suspension was plated at a density of 3 x 105 cells/cm2 on Millicell-CM inserts (0.4 µm pore size, 12 mm diameter, 0.6 cm2; Millipore Corporation, Bedford, MA, USA) coated with Vitrogen gel (0.15 mL/cm2, 0.24% collagen; Collagen Corporation, Palo Alto, CA, USA). The cell cultures were incubated at 37°C in a 5% CO2 atmosphere. Millicell inserts have a microporous membrane that allows access to both the apical and basolateral sides of the epithelial cells. Cell cultures were immersed in culture medium for 5 days, but without penicillin and streptomycin addition from day 1 onwards. After 5 days' incubation, they were switched to an airliquid interface by aspiration of the culture medium on the apical side and by applying medium via the basolateral side only. Characterization of the cell culture and adherence assays were performed on day 6 of culture.
Characterization of the cell culture on day 6
The number of cells per insert at confluence was determined as described previously.9 Trypan blue exclusion was used to check the viability of the cells.
For scanning electron microscopy the monolayers were fixed in glutaraldehyde 3% (Sigma-Aldrich) in 0.1 M sodium cacodylate buffer (pH 7.4) for 2 h. Tissue samples were dehydrated in graded ethanol series and dehydration was completed by critical-point drying with CO2. The mounted specimens were sputter-coated with gold and viewed in a PSEM 500 scanning electron microscope (Philips, Eindhoven, The Netherlands).
For transmission electron microscopy, the monolayers were fixed in glutaraldehyde 2.5% in 0.1 M phosphate buffer (pH 7.2) for several hours at 4°C, followed by post-fixation in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) for 1 h. The material was dehydrated in a graded ethanol series and then embedded in Epon 812. Ultrathin sections were made, stained with uranyl acetate and lead citrate and examined in an EM 10 transmission electron microscope (Carl Zeiss, Thornwood, NY, USA).
The ciliary beat frequency (CBF) was measured using computerized microscopic photometry.10 CBF recordings were taken from five different cells, with 10 consecutive measurements on each cell. Cell samples were placed in 0.3 mL of medium in a 24-well plate. Measurements were made at room temperature (20°C). Variability between cells was calculated from the standard deviations of the mean CBF of the five different cells from each measurement, and variability within cells was calculated from the standard deviations of the 10 consecutive CBF measurements from each cell.10
The expression of various cytokeratins in the cell layer was studied using an in situ immunohistochemical technique on frozen sections, using monoclonal antibodies directed against cytokeratin 7 (CK 7; Dako A/S, Glostrup, Denmark), CK 8 (Amersham International, Buckinghamshire, UK), CK 13 (Euro-Diagnostica AB, Malmö, Sweden), CK 14 (Sigma-Aldrich), CK 18 (Dako) and CK 19 (Amersham).9
Adhesion assays
From an overnight culture of S. pneumoniae on tryptic soy plates with 1% agar on to which 6300 U of catalase had been spread, 30 colonies (7080% transparent forms) were inoculated into 10 mL of brainheart infusion broth.11 After 4.5 h incubation at 37°C in 5% CO2 (mid-logarithmic phase), cultures were resuspended in culture medium (without penicillin and streptomycin) supplemented with 0.003% haemin (Sigma-Aldrich) and diluted to 107 cfu/mL. One hundred microlitres of this suspension was added to the inserts on day 6 of culture, and the inserts were further incubated for 4 h or 24 h at 37°C in 5% CO2. After incubation, each insert was washed three times in phosphate-buffered saline pH 7.4 to remove the non-adherent bacteria. The Vitrogen gel was removed by digestion with 400 µL collagenase (Wortington Biochemical Corporation, Freehold, NJ, USA) of a 200 IU/mL solution and then treated with 1500 µL trypsinEDTA (Gibco) to obtain a cell suspension. Epithelial cells were then lysed by addition of 1000 µL of 0.3% Triton X-100 (Roche Diagnostics, Brussels, Belgium). Adherent bacteria and non-adherent bacteria (bacteria in washing fluid) were quantified by plating appropriate dilutions on blood agar. Adherence was expressed as the percentage of the total cfu of bacteria per insert.
Transepithelial electrical resistance
The transepithelial electrical resistance (TEER) of the epithelial cell layer was measured using the Millicell electrical resistance system (Millipore). The observed TEER was corrected for the resistance of the blank filter (coated with Vitrogen gel).
To study the effect of pneumococci on the TEER of the cell layer, bacterial suspensions were prepared as for the adhesion assays, except that 450 µL of suspension, rather than 100 µL, was added to the inserts. The effect of lysis of the pneumococci on the TEER was studied by adding 50 µL of a penicillinstreptomycin solution (Gibco; final concentrations: penicillin 33 mg/L, streptomycin 56 mg/L) 3.5 h after inoculating the cell layer with pneumococci (6.3 x 104 cfu/insert).
Inhibitory effect of erythromycin
The cell cultures were first incubated for 18 h with culture medium containing a subinhibitory concentration (7.3 mg/L, 105 M) of erythromycin. The medium was then removed and erythromycin-containing fresh medium, with or without pneumococci (or IL-8 inducer in the IL-8 measurement experiments), was added. Adhesion assays were performed as described above for three different cell cultures, each performed in triplicate. The TEER of the cell layer was measured 0, 4, 7, 17, 19 and 24 h after inoculation. IL-8 was measured after 24 h incubation.
Determination of IL-8 secretion
The IL-8 concentrations in the culture supernatants were measured by a classical sandwich ELISA.12 The amount of IL-8 was expressed as ng/mL supernatant. Interleukin-1ß (IL-1ß, 100 U/mL) and lipopolysaccharide (LPS) from Escherichia coli O111/B4 (100 mg/L; Difco Laboratories, Detroit, MI, USA), known inducers of IL-8 secretion in epithelial cells, were used as controls.13
Assay for pneumolysin activity
Suspensions of pneumococci in cell culture medium (1:1 DMEMHam's F12 with high glucose supplemented with 2% Ultroser G and cholera toxin) with and without erythromycin (7.3 mg/L) were prepared as described in the section entitled Adhesion assays. The suspensions were incubated at 37°C in 5% CO2 for 40 h. Haemolytic activity (using fresh horse red blood cells washed three times) was measured as described by Paton et al.14 after 0, 10, 13, 16, 19, 23, 35 and 40 h of incubation. Viable counts (cfu/mL) were determined at the same time intervals by plating appropriate dilutions on to blood agar plates.
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Results |
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Within 6 days, cells grew to a confluent monolayer, which included ciliated cells with actively beating cilia. Of 1500 cells counted visually by light microscopy on three different inserts and for two different patients, 23.7% ± 2.5% were ciliated. Scanning electron microscopy revealed a confluent cell layer of ciliated cells and cells bearing microvilli. Transmission electron microscopy showed columnar epithelial cells, sometimes with a pseudostratified appearance. The nucleus was located basally. Ciliated cells were seen, with basal bodies present underneath the apical membrane. Other cells had microvilli and a glycocalyx. Junctional complexes between the cells were found near the apical membrane.
The average number of cells per insert, determined on five inserts, was 122600 ± 8764. The mean CBF from five different cells was 7.1 Hz. The between-cell and within-cell variability was 0.5 and 0.3 Hz, respectively.
The cell layer showed uniform cytoplasmic expression of CK 19. Only the suprabasal cells were uniformly positive for CK 7 and CK 18 and showed a focal staining pattern for CK 8. Faint and focal staining with CK 13 was observed in suprabasal cells. The cell layer did not express CK 14. This cytokeratin staining pattern is similar to that of normal respiratory epithelium.9
Effect of erythromycin on pneumococcal adherence
The effect of erythromycin on the adherence of pneumococci is shown in Figure 1. The adherence of pneumococci was significantly lower in the presence of subinhibitory concentrations of erythromycin, after both 4 h and 24 h. After 4 h, the mean ± S.E.M. percentage of pneumococci that had adhered to the epithelial cells was 4.0% ± 0.7% in medium with erythromycin compared with 7.7% ± 0.8% in medium without erythromycin (P = 0.002; Wilcoxon MannWhitney test); after 24 h, the corresponding values were 24.2% ± 5.3% and 38.4% ± 5.0%, respectively (P = 0.038). There was only a small (statistically not significant) difference in the total cfu recovered in the presence or absence of erythromycin: 3.0 x 107 ± 5.0 x 106 cfu were recovered in the absence of erythromycin compared with 2.0 x 107 ± 7.8 x 106 cfu (P = 0.43) in the presence of erythromycin after 4 h incubation and 4.4 x 107 ± 2.2 x 107 cfu compared with 3.9 x 107 ± 1.2 x 107 cfu (P = 0.166) after 24 h incubation. There was a 3.7- to 7.1-fold increase in adherence after 24 h incubation compared with 4 h incubation, with more variation between experiments in longer incubation. The viability of the epithelial cells after 24 h incubation with pneumococci was >90% both in the presence and in the absence of erythromycin.
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The TEERs of all cell layers varied between 50 and 1000 cm2 on day 6 of culture. Addition of erythromycin to the culture medium did not in itself affect the TEER of the cell layer.
Lysis of the pneumococci by high concentrations of penicillin caused an immediate decrease in the TEER, which continued for c. 6 h (Figure 2). For the next 10 h, the TEER remained relatively constant, but increased again afterwards.
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Cultured cells secreted 38.4 ± 5.9 ng/mL IL-8 constitutively. Stimulation of the epithelial cells with LPS and IL-1ß resulted in two-fold (78.3 ± 25.9 ng/mL) and three-fold (118.3 ± 17.4 ng/mL) increases in IL-8 secretion, respectively. Addition of pneumococci did not affect IL-8 secretion by the cells (45.7 ± 8.6 ng/mL). Addition of erythromycin to the culture medium did not significantly affect IL-8 secretion, either constitutive (32.2 ± 6.4 ng/mL) or induced (52.3 ± 3.8 ng/mL with LPS, and 116.3 ± 41.8 ng/mL with IL-1), by the cells. Addition of erythromycin to the culture medium in the presence of pneumococci did not affect IL-8 secretion.
Effect of erythromycin on pneumolysin release
In a pneumococcal suspension in culture medium without erythromycin, most pneumolysin was released after 1016 h of incubation (pneumolysin was assayed by measuring haemolytic activity); the release of pneumolysin was associated with a sharp decrease in viable counts (Figure 4). In the presence of subinhibitory concentrations of erythromycin, no increase in haemolytic activity was detected during the incubation time (40 h). The decrease in viable counts was less pronounced in the presence of erythromycin. Addition of erythromycin to culture medium in which pneumolysin release had already occurred did not reduce the haemolytic activity of pneumolysin (data not shown).
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Discussion |
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Starting from human nasal tissue, we obtained a confluent cell layer within 6 days of culture. The presence of tight junctions was confirmed by transmission electron microscopy and by the high TEER of the culture.21 Light and scanning electron microscopy showed that 2025% of the cells were ciliated. The CBF measurements (mean 7.1 Hz) are indicative of normal ciliary activity in our in vitro model.10 We used cytokeratin profiles as indicators of cell differentiation. The cytokeratin profile of our 6 day old cell layer was similar to that of a pseudostratified columnar epithelium except for the faint and focal staining for CK 13, which may suggest a change to a transitional pseudostratified epithelium in some areas.22
Study of pneumococcal adherence in the presence of erythromycin in our model revealed that subinhibitory concentrations of erythromycin led to a decrease in the adherence of S. pneumoniae to the epithelial cells. There are several possible explanations for this observation. (i) Erythromycin could reduce the expression of bacterial adhesion molecules. Erythromycin and azithromycin decrease piliation in P. aeruginosa and Neisseria gonorrhoeae.23 Adherence of pneumococci to human cells is achieved via surface proteins that bind eukaryotic carbohydrate receptors. The adhesin CbpA and the disaccharide N-acetylglucosamine ß13 galactose are known to participate in nasopharyngeal colonization in pneumococci.16,24 (ii) Erythromycin could inhibit the production of epithelial cell surface receptors, since erythromycin reduces respiratory glycoconjugate secretion.5 (iii) Since damaged epithelium is the preferred site for pneumococcal adherence,18 reduction in the amount of epithelial damage is also likely to reduce adherence. The smaller decrease in the TEER of the cell layer during the first 17 h after inoculation with pneumococci in the presence of erythromycin is an indication that the amount of epithelial disruption is reduced. Feldman et al.25 showed that pneumolysin alone was responsible for the epithelial disruption caused by pneumococcal filtrates. Pneumolysin, a major pneumococcal cytolytic toxin, is not actively secreted during bacterial growth. Release of pneumolysin is coupled to autolysis.26 We found that subinhibitory concentrations of erythromycin almost completely prevented the release of pneumolysin, which may explain the reduction in epithelial damage.
In our experiments, constitutive and stimulated IL-8 secretion by the cells was not significantly lower in the presence of erythromycin, in contrast to other reports.4,27 From our results it seems that inhibition of cytokine secretion in respiratory epithelial cells cannot explain the anti-inflammatory effects of erythromycin.
In conclusion, subinhibitory concentrations of erythromycin decrease the adherence of S. pneumoniae to respiratory epithelial cells in vitro, and thus interfere with a critical step in the pathogenesis of pneumococcal infection.
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Acknowledgments |
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Notes |
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References |
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2 . Goswami, S. K., Kivity, S. & Marom, Z. (1990). Erythromycin inhibits respiratory glycoconjugate secretion from human airways in vitro. American Review of Respiratory Disease 141, 728.[ISI][Medline]
3 . Takeyama, K., Tamaoki, J., Chiyotani, A., Tagaya, E. & Konno, K. (1993). Effect of macrolide antibiotics on ciliary motility in rabbit airway epithelium in vitro. Journal of Pharmacy and Pharmacology 45, 7568.[ISI][Medline]
4 . Suzuki, H., Shimomura, A., Ikeda, K., Furukawa, M., Oshima, T. & Takasaka, T. (1997). Inhibitory effect of macrolides on interleukin-8 secretion from cultured human nasal epithelial cells. Laryngoscope 107, 16616.[ISI][Medline]
5 . Ishida, L. K., Ikeda, K., Tanno, N., Takasaka, T., Nishioka, K. & Tanno, Y. (1995). Erythromycin inhibits adhesion of Pseudomonas aeruginosa and Branhamella catarrhalis to human nasal epithelial cells. American Journal of Rhinology 9, 535.[ISI]
6
.
Lagrou, K., Peetermans, W. E., Verhaegen, J., Van Lierde, S., Verbiest, L. & Van Eldere, J. (2000). Macrolide resistance in Belgian Streptococcus pneumoniae. Journal of Antimicrobial Chemotherapy 45, 11921.
7 . Jorissen, M., Van der Schueren, B., Van den Berghe, H. & Cassiman, J. J. (1989). The preservation and regeneration of cilia on human nasal epithelial cells cultured in vitro. Archives of Otorhinolaryngology 246, 30814.
8
.
Yamaya, M., Finkbeiner, W. E., Chun, S. Y. & Widdicombe, J. H. (1992). Differentiated structure and function of cultures from human tracheal epithelium. American Journal of Physiology 262, L71324.
9 . Hoefnagels-Schuermans, A., Peetermans, W. E., Jorissen, M., Van Lierde, S., Van Den Oord, J., De Vos, R. et al. (1999). Staphylococcus aureus adherence to nasal epithelial cells in a physiological in vitro model. In Vitro Cellular and Developmental Biology, Animal 35, 47280.[Medline]
10 . Jorissen, M. & Bessems, A. (1995). Normal ciliary beat frequency after ciliogenesis in nasal epithelial cells cultured sequentially as monolayer and in suspension. Acta Oto-Laryngologica 115, 6670.[ISI][Medline]
11 . Weiser, J. N., Austrian, R., Sreenivasan, P. K. & Masure, H. R. (1994). Phase variation in pneumococcal opacity: relationship between colonial morphology and nasopharyngeal colonization. Infection and Immunity 62, 25829.[Abstract]
12
.
Wuyts, A., Govaerts, C., Struyf, S., Lenaerts, J.-P., Put, W., Conings, R. et al. (1999). Isolation of the CXC chemokines ENA-78, GRO and GRO
from tumor cells and leukocytes reveals NH2- terminal heterogeneity. European Journal of Biochemistry 260, 4219.
13
.
Van Damme, J., Van Beeumen, J., Decock, B., Van Snick, J., De Ley, M. & Billiau, A. (1988). Separation and comparison of two monokines with lymphocyte-activating factor activity: IL-1ß and hybridoma growth factor (HGF). Identification of leukocyte-derived HGF and IL-6. Journal of Immunology 140, 153441.
14 . Paton, J. C., Lock, R. A. & Hansman, D. J. (1983). Effect of immunization with pneumolysin on survival time of mice challenged with Streptococcus pneumoniae. Infection and Immunity 40, 54852.[ISI][Medline]
15
.
Adamou, J. E., Wizemann, T. M., Barren, P. & Langermann, S. (1998). Adherence of Streptococcus pneumoniae to human bronchial epithelial cells (BEAS-2B). Infection and Immunity 66, 8202.
16 . Andersson, B., Dahmen, J., Frejd, T., Leffler, H., Magnusson, G., Noori, G. et al. (1983). Identification of an active disaccharide unit of a glycoconjugate receptor for pneumococci attaching to human pharyngeal epithelial cells. Journal of Experimental Medicine 158, 55970.[Abstract]
17 . Cundell, D. R., Weiser, J. N., Shen, J., Young, A. & Tuomanen, E. I. (1995). Relationship between colonial morphology and adherence of Streptococcus pneumoniae. Infection and Immunity 63, 75761.[Abstract]
18 . Rayner, C. F., Jackson, A. D., Rutman, A., Dewar, A., Mitchell, T. J., Andrew, P. W. et al. (1995). Interaction of pneumolysinsufficient and -deficient isogenic variants of Streptococcus pneumoniae with human respiratory mucosa. Infection and Immunity 63, 4427.[Abstract]
19 . Talbot, U. M., Paton, A. W. & Paton, J. C. (1996). Uptake of Streptococcus pneumoniae by respiratory epithelial cells. Infection and Immunity 64, 37727.[Abstract]
20 . Werner, U. & Kissel, T. (1996). In-vitro cell culture models of the nasal epithelium: a comparative histochemical investigation of their suitability for drug transport studies. Pharmaceutical Research 13, 97888.[ISI][Medline]
21 . Cereijido, M., Gonzalez Mariscal, L. & Contreras, R. G. (1988). Epithelial tight junctions. American Review of Respiratory Disease 138, S1721.[ISI][Medline]
22 . Stosiek, P., Kasper, M. & Moll, R. (1992). Changes in cytokeratin expression accompany squamous metaplasia of the human respiratory epithelium. Virchows Archiv A: Pathological Anatomy and Histopathology 421, 13341.[ISI]
23 . Yamasaki, T., Ichimiya, T., Hirai, K., Hiramatsu, K. & Nasu, M. (1997). Effect of antimicrobial agents on the piliation of Pseudomonas aeruginosa and adherence to mouse tracheal epithelium. Journal of Chemotherapy 9, 327.[ISI][Medline]
24 . Rosenow, C., Ryan, P., Weiser, J. N., Johnson, S., Fontan, P., Ortqvist, A. et al. (1997). Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae. Molecular Microbiology 25, 81929.[ISI][Medline]
25 . Feldman, C., Mitchell, T. J., Andrew, P. W., Boulnois, G. J., Read, R. C., Todd, H. C. et al. (1990). The effect of Streptococcus pneumoniae pneumolysin on human respiratory epithelium in vitro. Microbial Pathogenesis 9, 27584.[ISI][Medline]
26 . Rubins, J. B. & Janoff, E. N. (1998). Pneumolysin: a multifunctional pneumococcal virulence factor. Journal of Laboratory and Clinical Medicine 131, 217.[ISI][Medline]
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Khair, O. A., Devalia, J. L., Abdelaziz, M. M., Sapsford, R. J. & Davies, R. J. (1995). Effect of erythromycin on Haemophilus influenzae endotoxin-induced release of IL-6, IL-8 and sICAM-1 by cultured human bronchial epithelial cells. European Respiratory Journal 8, 14517.
Received 20 March 2000; returned 7 June 2000; revised 14 July 2000; accepted 24 July 2000