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Published ahead of print on April 24, 2003, doi:10.1165/rcmb.2002-0291OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 344-351, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0291OC

Induction of Regulated upon Activation, Normal T Cells Expressed and Secreted (RANTES) and Transforming Growth Factor-ß1 in Airway Epithelial Cells by Mycoplasma pneumoniae

Azzeddine Dakhama, Monica Kraft, Richard J. Martin and Erwin W. Gelfand

Program in Cell Biology, Departments of Pediatrics and Medicine, National Jewish Medical and Research Center, Denver, Colorado

Address correspondence to: Azzeddine Dakhama, Ph.D., Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail: dakhamaa{at}njc.org


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mycoplasma pneumoniae infection exacerbates asthma in children and may play a role in the pathogenesis of chronic asthma. Because the airway epithelium is a preferential site for M. pneumoniae infection and a major source of the chemokine regulated on activation, normal T cells expressed and secreted (RANTES) and transforming growth factor (TGF)-ß1, we postulated that this microorganism may contribute to the disease by inducing these mediators through direct interaction with airway epithelial cells. We investigated the effects of M. pneumoniae on RANTES and TGF-ß1 production in primary cultures of normal human bronchial epithelial (NHBE) cells and small airway epithelial (SAEC) cells. Both cell types were permissive to M. pneumoniae infection in vitro, but their responses were different. TGF-ß1 was induced at higher levels in NHBE than in SAEC cultures, whereas RANTES was induced in SAEC cultures but not in NHBE cultures. These effects were attenuated by erythromycin and dexamethasone. In vitro adherence assays further indicated that the effects of erythromycin were mediated through its antimicrobial action, resulting in diminished adherence of the pathogen, whereas the effects of dexamethasone did not appear to be by inhibition of adherence. These results suggest that M. pneumoniae infection may contribute to the pathogenesis of chronic asthma at different levels of the airways, by inducing TGF-ß1 in large airways and the chemokine RANTES in small airways.

Abbreviations: colony-forming units, cfu • enzyme-linked immunosorbent assay, ELISA • Hanks' balanced salt solution, HBSS • N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, HEPES • normal human bronchial epithelial cells, NHBE • optical density, O.D. • phosphate-buffered saline, PBS • regulated upon activation, normal T cell expressed and secreted, RANTES • reverse transcription and polymerase chain reaction, RT-PCR • small airway epithelial cells, SAEC • Tris-buffered saline, TBS • transforming growth factor-ß1, TGF-ß1 • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mycoplasma pneumoniae is a significant cause of tracheobronchitis, pharyngitis, and atypical pneumonia in humans (1). Because of its ability to infect and colonize the lower respiratory tract, the microorganism is considered an important pathogen in acute respiratory illnesses in children (2) and in adults with chronic airway diseases, such as asthma (3, 4) and chronic obstructive pulmonary disease (5). Support for the involvement of M. pneumoniae in asthma is shown by the ability of the pathogen to trigger Th2-type cytokines and IgE responses (6, 7), to activate mast cells (8), to result in deterioration of pulmonary function (9, 10), and potentially to alter the pharmacologic control of the disease.

The ciliated airway epithelium is the primary site of M. pneumoniae infection in the lungs where sialo-oligosaccharide receptors, present in both large and small airways, are polarized at the cilia and microvillar domains of epithelial cells (11). However, it is unknown whether distinct patterns of airway responses may be produced in different locations of the respiratory tract following M. pneumoniae infection. The epithelium is a major source of a variety of mediators, including cytokines and chemokines, which have the potential to recruit and activate inflammatory cells at the site of infection. We hypothesized that the epithelium could play a primary role in modulating the local airway response to M. pneumoniae infection through release of cytokines and chemokines that may contribute to the pathogenesis of chronic asthma. The present study was performed to investigate the effects of M. pneumoniae infection on the function of normal human, large and small airway epithelial cells in primary cultures in vitro. More specifically we focused our attention on the profibrosis cytokine transforming growth factor (TGF)-ß1 and the CC chemokine regulated on activation, normal T cells expressed and secreted (RANTES), two important mediators that may play significant roles in asthma. The results demonstrated that both large (bronchial) and small human airway epithelial cells were permissive to M. pneumoniae infection in vitro; however, their responses to the pathogen were distinguishable. Whereas TGF-ß1 was induced predominantly in large airway epithelial cells, RANTES production was only induced in small airway epithelial cells. Dexamethasone inhibited the production of both TGF-ß1 and RANTES, but did not alter the adherence of M. pneumoniae to epithelial cells, whereas erythromycin-mediated similar inhibitory effects essentially by reducing the amounts of adhered pathogen.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Epithelial Cell Culture
Normal human bronchial epithelial (NHBE) and small airway epithelial (SAEC) cells were purchased from Clonetics (Walkersville, MD) and used at culture passages 3–5. The cells were grown in serum-free bronchial epithelial cell growth medium (BEGM; Clonetics) containing the following supplements (all from Clonetics): bovine pituitary extract (52 µg/ml), hydrocortisone (0.5 µg/ml), human epidermal growth factor (0.5 ng/ml), epinephrine (0.5 µg/ml), transferrin (10 µg/ml), insulin (5 µg/ml), retinoic acid (0.1 ng/ml), triiodothyronine (6.5 ng/ml), gentamycin (50 µg/ml), and amphotericin B (50 ng/ml). When the cultures reached 70–80% of confluence, the cells were detached by incubation with 0.1 M N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid (Hepes)-buffered Hanks' balanced salt solution (HBSS; Clonetics), pH 7.2, containing 0.025% trypsin and 0.01% ethylenediaminetetraacetic acid (EDTA; Clonetics) and passaged into new culture plates or flasks.

Mycoplasma Culture and Epithelial Cell Infection
M. pneumoniae, strain 15531, was obtained from American Type Culture Collection (ATCC, Manassas, VA) and was grown at 37°C in PPLO medium (DIFCO Laboratories, Detroit, MI) supplemented with 10% horse serum (DIFCO), penicillin (100 U/ml), and polymyxin (500 U/ml). Actively growing cultures were harvested on Day 4, by collecting both adherent and nonadherent microorganisms, and centrifuged at 10,000 x g for 15 min at 10°C. The resulting pellets were re-suspended in Hepes-buffered HBSS, pH 7.4, and washed twice. The concentration of M. pneumoniae (colony-forming units [cfu]/ml) was determined from a pre-established standard curve by reading of optical density (O.D., {lambda} = 660 nm) where 1 O.D. unit corresponds to 1.3 x 109 viable cfu/ml (12). In some experiments, nonviable M. pneumoniae preparations were used as control and were obtained by subjecting the suspensions either to 15 freeze-thaw cycles or to ethanol fixation (15 min), which resulted in killing of the pathogen as determined by culture on PPLO agar plates (cat# 20260; REMEL, Lenexas, KS). For infection with viable bacteria, the suspension of freshly harvested M. pneumoniae was adjusted to {bsim} 108 cfu/ml and further diluted with supplement-free BEGM medium to obtain a designated infectious dose of 10–100 cfu/cell. Hydrocortisone and growth factors were withdrawn from the cultures 12 h before infection, to avoid potential interference by effects from these media supplements.

Assessment of Endotoxin Contamination and Effects on Epithelial Cells
Endotoxin contamination was assessed in M. pneumoniae preparations and cell cultures by using a Limulus Amoebocyte Lysate (LAL)-based Pyrogent Plus Gel Clot assay with a sensitivity of 0.06 endotoxin units/ml (Cat # N283–06; BioWhittaker, Inc., Walkersville, MD). To assess possible direct effects (if any) of endotoxin on TGF-ß1 and RANTES expression by epithelial cells, some cultures were incubated with lipopolysaccharide from Escherichia coli 055:B5 (Sigma, St. Louis, MO) at a concentration of 1 µg/ml, with a corresponding endotoxin activity of 8,000 U/ml determined by LAL assay as described above.

Detection of M. pneumoniae
After infection, the presence of M. pneumoniae in epithelial cell cultures was monitored at the cellular level by immunoperoxidase using a rabbit polyclonal antibody (kindly provided by Dr. H. W. Chu, National Jewish Medical and Research Center, Denver, CO) and confirmed by polymerase chain reaction (PCR) detection using oligonucleotide primers specific for the 3' flanking region of M. pneumoniae 16S ribosomal RNA gene (Table 1). For immunocytochemistry, epithelial cells were grown in sterile 8-chamber tissue culture treated glass slides (Becton Dickinson Labware, Franklin Lakes, NJ), infected with M. pneumoniae, then washed and fixed for 10 min with methanol. After rehydration, the cells were incubated for 15 min with H2O2 (0.3% in 0.1% NaN3), to block endogenous peroxidase, followed by 15-min incubation with 1% normal goat serum (DAKO Corp., Carpinteria, CA) to prevent nonspecific binding of secondary antibody. Cells were incubated for 2 h at room temperature with rabbit polyclonal antibody to M. pneumoniae, optimally diluted 1:2,000 in 50 mM Tris-buffered saline (TBS), pH 7.6, containing 1% bovine serum albumin and 0.05% Tween-20. After 3 washes with TBS, the cells were incubated for 30 min with a biotinylated goat anti-rabbit immunoglobulin (DAKO) followed by washing and 30-min incubation with peroxidase-conjugated avidin-biotin (ABC) complex (DAKO). After washing, the reaction was developed by incubation with Fast-DAB peroxidase substrate (Sigma) followed by counterstaining of nuclei with Harris's hematoxylin. Controls for immunostaining consisted of M. pneumoniae–infected cells incubated with normal rabbit serum, which resulted in no staining (not shown), and uninfected cells incubated with rabbit anti–M. pneumoniae antibody.


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  		TABLE 1 Oligonucleotide PCR primers

 
In Vitro M. pneumoniae Adherence Assay
Epithelial cells were seeded at 5,000 cells/well in 96-well tissue culture plates and grown for 3 d. Just before inoculation, duplicate culture wells were selected from the center and from each corner of the plate for cell counting purposes only. The number of cells grown in these wells was then used to adjust the infectious dose (cfu/ml) for all other wells. For adherence assays, the culture medium was aspirated in each well and replaced by 50 µl of M. pneumoniae suspension (adjusted with culture medium to obtain a designated infectious dose). The inoculated cultures were incubated at 37°C for 6 h to allow for maximal adherence of M. pneumoniae (determined from preliminary experiments, data not shown) followed by five consecutive washes with HEPES-buffered HBSS and addition of fresh medium for an additional period of culture. To test the effects of dexamethasone and erythromycin on M. pneumoniae adherence, the drugs were added at the time of infection and were maintained in the culture thereafter. After culture, the wells were washed as before and fixed in cold methanol for 10 min. After rehydration with distilled water followed by 10 mM phosphate-buffered saline (PBS), pH 7.4, the cells were incubated with polyclonal rabbit anti–M. pneumoniae antibody (1:4,000) for 2 h at room temperature and washed with PBS. Subsequent steps consisted of serial incubations with a biotinylated goat anti-rabbit antibody (diluted 1:1,000, 60 min; DAKO) followed by avidin-conjugated peroxidase (diluted 1:500, 30 min; Sigma) and soluble TMB peroxidase substrate (BD Pharmingen, San Diego, CA). The reaction was stopped by addition of 2 N H2SO4. The amounts of adhered M. pneumoniae were determined by measuring the absorbance at 450 nm after subtracting the background values from similarly stained, uninfected cell culture wells.

Culture Isolation of Viable M. pneumoniae
To assess the viability of M. pneumoniae in the cultures following infection and treatment with dexamethasone (1 µM) or erythromycin (50 µg/ml), both epithelial cells and culture supernatants were examined for the presence of viable M. pneumoniae by culture isolation using selective Mycoplasma PPLO agar medium (REMEL). Briefly, NHBE and SAEC cultures were grown to {bsim} 75% confluence in 24-well culture plates and inoculated with M. pneumoniae at a dose of 20 cfu/cell. After 6 h of incubation, the cells were gently washed three times followed by incubation with 500 µl of supplement-free culture medium for additional periods of culture. At different time points (24, 48, and 96 h) following infection, supernatants were harvested and cells were detached from each well by incubation for 2 min at room temperature with 250 µl of Trypsin/EDTA solution (Clonetics) followed by 250 µl of Trypsin Neutralizing solution (Clonetics). In preliminary assays, this method produced better results than cell scraping ({bsim} 2-fold higher yield) for isolating epithelial cell-associated viable M. pneumoniae. For M. pneumoniae culture isolation, 50-µl aliquots of recovered cells or culture supernatants were initially diluted 1:10 with PPLO broth medium (DIFCO laboratories). The suspensions were first pre-incubated at 37°C for 2 h to help release the microorganisms into the medium, then serial decimal dilutions were made followed by plating of 10 µl of each dilution onto PPLO agar plates using sterile plating glass rods. The plates were incubated at 37°C for 3–4 wk until the appearance of colonies of M. pneumoniae typically characterized by "fried egg" morphology.

Analysis of Messenger RNA Expression
Total RNA was extracted from cells by using RNeasy total RNA extraction kit following manufacturer's instruction (Qiagen, Valencia, CA). Amplification by reverse transcription and polymerase chain reaction (RT-PCR) was performed as previously described (13). Briefly, 1 µg of extracted total RNA was linearized and transcribed into cDNA for 30 min at 42°C using MMLV reverse transcriptase and oligo-dT12–18 primer (GIBCO-BRL, Rockville, MD). Aliqouts of transcribed cDNA (100 ng total RNA equivalent) were amplified by 35 cycles of PCR using Taq DNA polymerase (GIBCO-BRL) and specific oligonucleotide primers (Table 1). RT-PCR products were resolved by electrophoresis on ethidium bromide–stained agarose gels and visualized by ultraviolet light illumination. The relative mRNA abundance was determined by measuring target:ß-actin mRNA ratio using NIH Scion Image analysis program (version 1.62, developed at the US National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). Positive RT-PCR controls were obtained by amplification of mRNA from cultures stimulated with recombinant human tumor necrosis factor (TNF)-{alpha} (0.5 ng/ml; R&D Systems Inc., Minneapolis, MN).

Cytokine Measurements
The levels of RANTES were measured in culture supernatants using an enzyme-linked immunosorbent assay (ELISA) kit following the manufacturer's instructions (R&D Systems). The levels of TGF-ß1 were determined by ELISA according to the manufacturer's instructions (BD Pharmingen), after acidification of culture supernatants, to detect both the latent and active forms of TGF-ß1, or without acidification to detect the active form only. The sensitivities of the assays were 2.9 pg/ml for RANTES and 30 pg/ml for TGF-ß1.

Statistical Analysis of Data
All cultures and treatments were performed in quadruplicate experiments. Data are presented as mean ± SEM. Data were analyzed for statistical significance with a P value of < 0.05 by ANOVA with Bonferroni correction for multiple comparisons of the means.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As detected by immunoperoxidase staining, M. pneumoniae was persistently present on epithelial cells for up to 96 h after inoculation, and its presence in the cultures was further confirmed by PCR (Figure 1). No apparent cytopathic effects (cell detachment, rounding, or vacuolization) were observed in the infected cultures over the 96-h co-culture period, and there were no significant differences between NHBE and SAEC cultures in the proportion of infected cells. As detected by immunostaining, the proportion of M. pneumoniae–positive cells reached up to 80% in both cell type cultures depending on the infectious dose (Figure 2A). However, neither the proportion of infected cells nor the amounts of adhered M. pneumoniae increased after initial attachment of the pathogen, which was maximal after 6 h of incubation under the experimental conditions (Figures 2B and 2C). Together, these data indicated that both cell types were similarly permissive to M. pneumoniae infection and suggested that the pathogen did not replicate under these culture conditions.



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  		Figure 1. Detection of M. pneumoniae. NHBE and SAEC were grown in 8-chamber tissue culture treated glass slides and co-cultured with M. pneumoniae as described in MATERIALS AND METHODS. M. pneumoniae was detected by immunocytochemistry in both infected NHBE (B) and SAEC (D) cultures (arrows), but not in uninfected NHBE (A) and SAEC (C) cultures. Scale bar represents 50 µm. In duplicate cultures, the presence of M. pneumoniae was confirmed by PCR analysis (E: agarose gel showing specific PCR products for M. pneumoniae. Lane 1: M. pneumoniae control DNA; lane 2: uninfected NHBE; lane 3: infected NHBE; lane 4: uninfected SAEC; lane 5: infected SAEC).

 


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  		Figure 2. Dose- and time-related permissiveness of normal airway epithelial cells to M. pneumniae infection in vitro. (A) Proportion of M. pneumoniae-positive cells, detected by immunostaining at 24 h following inoculation with M. pneumoniae at varying multiplicity of infection (*significant difference when compared with infectious doses of 10). (B) Proportion of M. pneumoniae–positive cells detected by immunostaining at various time periods of culture following inoculation with M. pneumoniae at optimal infectious dose (20 cfu/cell, determined from dose–response experiments). (C) Time-related adherence of M. pneumoniae to airway epithelial cells in culture. Cells were incubated with M. pneumoniae (20 cfu/cell) for 6 h to allow for maximal adherence, then washed with medium and further cultured for the indicated time periods. Up to 80% of cells were positive by immunostaining for M. pneumoniae depending on the infectious dose (A). However, neither the proportion of infected cells nor the amounts of adhered M. pneumoniae increased in the cultures after the 6-h initial incubation (B, C). Black bars, NHBE; hatched bars, SAEC.

 
Figure 3 illustrates representative agarose gels showing the results of RT-PCR analysis for TGF-ß1 and RANTES mRNA expression in NHBE and SAEC cultures following M. pneumoniae infection and the effects of dexamethasone and erythromycin treatments (Figure 3A). The results revealed significant differences between the two cell type cultures in the abundance of TGF-ß1 and RANTES mRNA relative to ß-actin mRNA levels (Figure 3B). No TGF-ß1 or RANTES mRNA expression was detected in the cultures at 6 h after initial incubation with M. pneumoniae (data not shown). At 24 h after infection, TGF-ß1 mRNA was induced to higher levels in NHBE than in SAEC cultures, whereas RANTES mRNA was only induced in infected SAEC cultures. Treatment with either dexamethasone or erythromycin resulted in significantly lower levels of TGF-ß1 and RANTES mRNA expression in both infected cell cultures. Interestingly, although declining after 48 h of infection, the induced mRNA expression persisted in both cultures for 96 h.




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  		Figure 3. (A) Representative agarose gels showing RT-PCR products for TGF-ß1 and RANTES mRNA expressed by NHBE and SAEC cultures at 24 h following M. pneumoniae infection and the effects of treatment with dexamethasone or erythromycin. Lane 1: uninfected cultures (negative control); lane 2: TNF-stimulated cultures (positive control); lane 3: M. pneumoniae–infected cultures; lane 4: M. pneumoniae–fected, dexamethasone-treated cultures; lane 5: M. pneumoniae–infected, erythromycin-treated cultures. (B) Time-related changes in relative TGF-ß1 and RANTES mRNA abundance in NHBE and SAEC cultures following infection by M. pneumoniae in vitro. Data are presented as a ratio of target (TGF-ß1 and RANTES) mRNA to ß-actin mRNA signals. CTL, uninfected control cultures; Mp, infected cultures; Mp/Dex, infected and treated with dexamethasone; Mp/Em, infected and treated with erythromycin. TGF-ß1 was induced in both airway epithelial cells, at relatively higher levels in NHBE (black bars) compared with SAEC (hatched bars) cultures following M. pneumoniae infection in vitro. By contrast, RANTES was only induced in SAEC cultures. Both TGF-ß1 and RANTES were induced to near maximal expression levels by 24 h of infection. The expression levels declined after 48 h but persisted for up to 96 h following infection. At the peak of induced mRNA expression (24 h), both dexamethasone and erythromycin treatments resulted in significant inhibition of TGF-ß1 and RANTES expression (*significant difference compared with control; #significant difference compared with Mp).

 
Further analyses demonstrated that viable M. pneumoniae was required for the induction of these responses, which were not observed in both epithelial cell type cultures following incubation with the same infectious dose of microorganisms that were either killed by multiple freeze–thaw cycles or fixed with ethanol (data not shown). In addition, incubation of both types of epithelial cells for up to 96 h with lipopolysaccharide (1 µg/ml) failed to induce detectable mRNA expression for TGF-ß1 or RANTES, suggesting that the responses cannot be attributed to endotoxin contamination. Moreover, no endotoxin contamination was detected in the preparations of M. pneumoniae and the levels remained below the detection limits (< 0.06 U/ml) in supernatants collected at 24, 48, and 96 h from infected epithelial cell cultures, further indicating that M. pneumoniae is not a source of endotoxin.

At the protein level, TGF-ß1 was secreted from both infected epithelial cell types in a latent form (active TGF-ß1 was not detected), but the levels were significantly higher in NHBE than it was detected in SAEC cultures from 24–96 h after infection (Figure 4). By contrast, RANTES protein secretion was detected only in infected SAEC cultures at levels that gradually increased over the 96-h culture period. Both TGF-ß1 and RANTES secretions were significantly reduced by treatments with dexamethasone and erythromycin.



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  		Figure 4. Kinetics of TGF-ß1 and RANTES secretions in M. pneumoniae–infected NHBE and SAEC cultures and effects of treatments with dexamethasone and erythromycin (same legend as in Figure 3B). Only the latent form of TGF-ß1 was detected in the supernatants of both cell type cultures following infection. The levels were significantly higher in NHBE compared with SAEC cultures from 24–96 h after infection (P < 0.05). In both infected cultures, TGF-ß1 secretions were significantly inhibited by treatments with dexamethasone or erythromycin. RANTES secretions were detected only in infected SAEC cultures. The secretion levels increased progressively from 24 h to 96 h following infection and were significantly inhibited by dexamethasone and erythromycin treatments (*significant differences compared with Mp). Open circles, CTL; filled circles, Mp; open triangles, Mp/Dex; open squares, Mp/Em.

 
To further identify the mode of inhibitory action by dexamethasone and erythromycin on the responses to M. pneumoniae infection, an in vitro 96-well culture plate-based M. pneumoniae adherence assay was developed and tested. The results showed that treatment with erythromycin, but not dexamethasone, resulted in significantly reduced amounts of adhered M. pneumoniae in both NHBE and SAEC cultures (Figure 5). To determine if these effects were associated with a loss in viability of M. pneumoniae in the epithelial cell cultures, both culture supernatants and cells were harvested and cultured on PPLO agar medium enriched for selective isolation of viable M. pneumoniae. The results revealed that viable M. pneumoniae was mostly cell-associated and persisted in the cultures for up to 96 h after infection. Treatment of infected cultures with dexamethasone did not alter the viability of M. pneumoniae as most viable microorganisms remained cell-associated. By contrast, treatment with erythromycin resulted in markedly decreased numbers of cell-associated viable M. pneumoniae in the cultures with no release of viable free microorganism into the culture supernatants (Table 2).



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  		Figure 5. Effect of dexamethasone or erythromycin on the adherence of M. pneumoniae to airway epithelial cells. NHBE and SAEC were grown in 96-well culture plate and incubated with M. pneumoniae (20 cfu/cell) in the absence (control) or presence of varying doses of dexamethasone or erythromycin for 24 h of culture. Adherence of M. pneumoniae was evaluated by spectrophometric reading of optical density following detection by immunoperoxidase using a soluble substrate, as described in MATERIALS AND METHODS. The data are presented as percent of adherence relative to infected control cultures. Dexamethasone treatment did not alter the adherence of M. pneumoniae at any of the doses tested. By contrast, treatment with erythromycin resulted in significantly reduced amounts of adhered M. pneumoniae in both airway epithelial cell cultures (*significant differences between effects of treatments with 50 µg/ml and treatments with 1 or 10 µg/ml). Black bars, NHBE; hatched bars, SAEC.

 

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  		TABLE 2 Effects of dexamethasone and erythromycin on the viability of M. pneumoniae in NHBE and SAEC cultures

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The airway epithelium is an important structural and functional interface between the environment and the lung immune system. Pathogens that reach the lower respiratory tract have to first interact with airway epithelial cells, which respond by producing a variety of mediators (cytokines, chemokines, lipid mediators, growth factors etc.) to integrate both local, resident, and peripheral immune inflammatory systems to elicit appropriate responses. The present study demonstrates that both large (NHBE) and small (SAEC) airway epithelial cells are permissive to M. pneumoniae infection in vitro, but distinct responses can be elicited. Although NHBE cells produced significantly larger amounts of TGF-ß1 than SAEC, only the latter produced RANTES in response to Mp infection.

TGF-ß1 is an important regulator of tissue fibrosis, but also has anti-inflammatory properties (14). TGF-ß1 is naturally produced as an inactive, latent dimeric protein, which forms a complex with a latency-associated peptide. To mediate its biological effects, latent TGF-ß1 needs to be activated by cleavage and dissociation of latency-associated peptide, a process that can be induced by factors such as plasmin, cathepsin or thrombospondin (15–17). Active TGF-ß1 is chemotactic and mitogenic for fibroblasts and known to induce angiogenesis and fibrosis by increasing collagen synthesis (18) and by promoting the expression of collagenase and metalloproteinase inhibitors (19). TGF-ß1 levels are increased in the airways of individuals with asthma (20), and there is evidence to suggest that in addition to fibroblasts and infiltrating eosinophils, bronchial epithelial cells are also a source in vivo (21, 22). It is also thought that direct interactions between epithelial cells and myofibroblasts likely occur in vivo in asthmatic airways and may play a central role in airway fibrosis, conceivably as a result of an abnormal repair process following periodic injuries to and shedding of the epithelium (23). In an elegant study, in which adenovirus was used as a vector, Sime and coworkers induced considerable fibrosis in rat airways just by overexpressing the active, but not the latent, form of TGF-ß1 in airway epithelial cells (24). Thus, if epithelial cells are triggered as a source for TGF-ß1, they may play a direct role in the subepithelial fibrosis that develops in their immediate vicinity where myofibroblasts are thought to migrate and release newly synthesized collagen (25). However, the development of a true fibrotic response is further controlled by additional mechanisms involving interaction of active TGF-ß1 with its receptors expressed on target fibroblast cells and the expression of a family of TGF-ß1 signal transduction proteins, Smad, which regulate TGF-ß1–responsive genes (26). In the present study, TGF-ß1 was secreted in a latent form, which was detected by the ELISA assay only after activation by acidification of samples. Nonetheless, if not activated immediately after its secretion, latent TGF-ß1 can bind to the extracellular matrix and become available for activation during an inflammatory process such as in asthma, where profibrotic conditions are enhanced in the airways (27).

The airway epithelium is a considerable source of the CC chemokine RANTES, which attracts eosinophils, basophils, monocytes, and memory T lymphocytes, and thus may play a central role in asthma (28). RANTES was found to be expressed in the airways of patients with stable allergic asthma (29), and its levels were significantly increased after endobronchial allergen challenge (30). Recently, functional polymorphisms have been identified in the promoter region of the RANTES gene, which were associated with atopy and asthma in one study (31), and with the development of late-onset asthma in another (32). Of relevance to the present study was the differential effect of M. pneumoniae infection on RANTES production by small (SAEC) versus large (NHBE) airway epithelial cells. Although both epithelial cells showed similar permissiveness to M. pneumoniae infection in vitro, RANTES production was only induced in small airway epithelial cells. The reason for these differences is unknown, but it may be due to different intrinsic properties that characterize the response of these cells to exogenous stimuli. For example, TNF-{alpha} stimulation induced RANTES expression in both epithelial cell types, but the levels were higher in SAEC cultures (Figure 3). A recent study investigated the effects of M. pneumoniae on RANTES production by primary cultures of nasal epithelial cells established from children with and without asthma (33). The authors found that unlike viral pathogens such as respiratory syncytial virus, M. pneumoniae did not induce RANTES secretion by these cells. This finding is not inconsistent with ours, and further supports the notion that M. pneumoniae may induce distinct responses in different locations of the respiratory tract (from upper to lower, large versus small airways).

Glucocorticoids are the most effective anti-inflammatory therapeutic drug, commonly prescribed in asthma. It is also remarkable that prolonged macrolide antibiotic treatment may improve symptoms in patients with severe, corticosteroid-dependent asthma, including those with documented infection by atypical microorganisms (34, 35). Macrolides are the most active drugs against M. pneumoniae and Chlamydia pneumoniae. The present study demonstrated that in spite of attenuating RANTES and TGF-ß1 production by infected airway epithelial cells, dexamethasone treatment did not alter the adherence of M. pneumoniae, which remained attached on the surface of these cells. On the other hand, targeting M. pneumoniae with erythromycin, which reduced the viability of the pathogen and hence its adherence, resulted in significant inhibition of the production of both cytokines. These data suggest that, besides potential anti-inflammatory activity (36), the beneficial effects of macrolide treatment may primarily result from a direct antimicrobial effect resulting in reduced adherence to and colonization of the airway epithelium.

In summary, the results of this study suggest that M. pneumoniae infection may contribute to the pathogenesis of chronic asthma by inducing the CC chemokine RANTES and the profibrosis cytokine TGF-ß1 in airway epithelial cells. Based on the present in vitro observations, it is tempting to speculate that both the large and small airways may be involved in M. pneumoniae infection with distinct patterns of responses, one potentially leading to remodeling in the large airways, but with greater inflammation in the smaller airways. Further studies, in vivo, are needed to validate this hypothesis and to define possible differences that may exist between asthmatic and nonasthmatic airway epithelial cells in term of susceptibility and patterns of response to M. pneumoniae and medication.


   Acknowledgments
 
This work was supported by ALA ARC and in part by grants from the National Institutes of Health (HL-60015 and HL-36577) and Environmental Protection Agency (grant R825702).

Received in original form December 5, 2002

Received in final form April 1, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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