I. General introduction
4. AIMS OF THE STUDY
Alveolar macrophages, the most abundant immune cells in the alveolar spaces, are key players in maintaining homeostasis and regulating immune defenses in the lung.
However, the epithelial cells lining the airways are the first point of contact for inhaled pathogens. In recent years the role of epithelial cells in lung innate immunity has become a focus of research interest, as it was discovered that airway epithelial cells possess not only passive barrier function, but also actively contribute to fighting respiratory pathogens.
• The aim of the first part of the study was to establish an in vitro lung co-culture model to study the contribution of epithelial cells to inflammation upon stimulation with bacterial stimuli from Gram-positive and Gram-negative bacteria. In addition mechanisms of LPS-induced inflammatory modulation were characterized. The co-culture model was composed of the alveolar epithelial type II like A549 cell line and peripheral blood mononuclear cells (PBMC).
Recent in vivo investigations suggest that chronic exposure to environmental factors like LPS induces lung inflammation and subsequent chronic lung diseases, including pulmonary fibrosis. Myofibroblasts, the main cell population in pulmonary fibrosis, are central in the pathogenesis of this disease. It is thought that epithelial cells may differentiate into myofibroblasts through a process called epithelial to mesenchymal transition (EMT). Recent understandings in the molecular mechanisms of fibrosis show that EMT is the missing link between inflammation and fibrosis and indicate an important role of TGF-β1 as master switch for EMT induction. However, the role of LPS in the process of EMT was not studied so far.
• The aim of the second study was to investigate whether LPS stimulation of PBMC in vitro induces an inflammatory response that mediates EMT of lung epithelial A549 cells. A subsequent goal was to establish the identity of the cytokine(s) produced by PBMC which mediated EMT of A549 cells.
II.
Lung epithelial cells constitutively produce an immunomodulatory
factor for cytokine release by mononuclear cells
1. ABSTRACT
The contribution of airway epithelial cells to the innate immune response in the lung has been a focus of recent interest. A human mixed co-culture system of lung epithelial cell line A549 and primary peripheral blood mononuclear cells (PBMC) allowed us to study modulation of IL-1β, TNF-α, IL-6 and IL-8 expression triggered by lipopolysaccharide (LPS), staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN) in comparison to respective monocultures. All immune stimuli induced significantly less TNF-α and much higher IL-6 release in co-cultures compared to PBMC alone. IL-1β release was stimulus-dependent: down-regulated upon LPS, up-regulated upon PGN and not affected upon SEB stimulation. IL-8 amounts were significantly increased in LPS and PGN-stimulated co-cultures and slightly increased upon SEB stimulation. Transwell experiments showed that LPS-induced cytokine modulation in co-culture is triggered by soluble factors. Using neutralizing IL-1β antibody we demonstrated that PBMC-derived IL-1β mediates augmented IL-6 and IL-8 production in LPS-stimulated co-cultures. A still unknown soluble factor in conditioned supernatant of resting A549 cells was shown to down-regulate TNF-α release by PBMC on mRNA level. Characterization of this anti-inflammatory compound suggests a peptide nature and opens an interesting area of future research.
In conclusion, lung epithelial cells modulate the inflammatory response by down-regulating TNF-α and increasing IL-6 and IL-8 in co-cultures and must therefore be considered actors in shaping lung inflammation.
2. INTRODUCTION
The human lung is exposed to a large number of airborne pathogens as a result of the daily inhalation of 10 000 liters of air. The observation that respiratory infections are nevertheless rare is due to the presence of an efficient host defense system in the lung. The airway epithelium represents a primary site of entrance and deposition of potentially pathogenic microorganisms into the body, and therefore is equipped with a variety of mechanisms to avoid infections (Bals & Hiemstra, 2004), (Diamond et al., 2000). The epithelial lining fluid in the lower respiratory tract contains immune cells: alveolar macrophages, T- and B-cells, neutrophils, eosinophils, mast cells and dendritic cells. Alveolar macrophages, derived from blood monocyte differentiation, account for up to 95% of the cells recovered by bronchoalveolar lavage (Bingisser &
Holt, 2001) and play a critical role in maintaining homeostasis, host defense, response to foreign substances and tissue remodeling (Losa Garcia et al., 1999). To combat infection, the phagocytic cells of the innate immune system express pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) on the surface of microorganisms. Toll-like receptors (TLRs) function as major PRRs in mammals. TLRs are membrane-bound molecules expressed on the surface or within intracellular compartments that participate in the recognition of different microbial compounds. 10 members of this family (TLR 1-10) have been identified in humans so far. The most prominent PAMP, lipopolysaccharide (LPS or endotoxin) from the cell wall of gram-negative bacteria, is recognized by TLR-4 (Akira et al., 2001), (Takeda & Akira, 2005). Intracellular recognition of bacteria appears to also involve a TLR-independent system. Recent studies indicate that nucleotide-binding oligomerization domain (NOD) 1 and 2 proteins recognize peptidoglycan (PGN) present in the cell wall of Gram-positive bacteria (Mogensen, 2009). Staphyloccocal enterotoxin B (SEB, superantigen) acts by directly binding to major histocompatibility complex class II molecules on antigen-presenting cells and is recognized by αβ receptors on T-cells. Therefore, it is involved in activation of the adaptive immune system response (Choi et al., 1989), (Krakauer, 2001).
A number of studies indicated that TLRs are also expressed in airway epithelial cells (Gomez & Prince, 2008), (Muir et al., 2004). It was demonstrated that primary tracheo-bronchial epithelial cells (Becker et al., 2000), alveolar epithelial type II A549
and bronchial epithelial BEAS-2B cell lines (Schulz et al., 2002) express mRNA for TLRs 1-6. Studies on TLR-4 localization in A549 and BEAS-2B cell lines have described a perinuclear location in association with the Golgi apparatus, rather than at the cell surface. Despite TLR-4 expression these cell lines were not directly responsive to LPS in serum-free medium, rather in the presence of serum and concentrations higher the 1µg/ml (Guillot et al., 2004). Furthermore, it was observed that primary lung epithelial cells and cell lines, release 6 and 8 in response to IL-1β and TNF-α stimulation (Coulter et al., 1999), (Henriquet et al., 2007), (Jiang, Kunimoto & Patel, 1998). IL-1β and TNF-α, are the most important early responsive pro-inflammatory cytokines by immune cells in innate immune response (Strieter et al., 2002). Secretion of IL-8 chemokine during bacterial infections in the lung was a prerequisite for recruitment of neutrophils into the alveolar space (Reutershan & Ley, 2004). Apart of acute phase reactions, IL-6 was found to participate in modulation of lung immune responses exerting stimulus-dependent pro- and anti-inflammatory activities (Xing et al., 1998).
An in vitro co-culture lung model was devised to study the contribution of epithelial cells to inflammation in the lung. Here we worked with human pulmonary epithelial type II cells (A549) and primary peripheral blood mononuclear cells (PBMC). Our findings indicate a function of alveolar epithelial cells in modulating the inflammatory reaction in the lung.
3. RESULTS
3.1. A549 cells modulate inflammatory response of PBMC upon LPS, SEB and PGN stimulation
A549 cells directly stimulated with 100 ng/ml LPS, 100 ng/ml SEB or 1 µg/ml PGN did not release IL-1β, TNF-α, IL-6 or IL-8 (data not shown). However, the presence of epithelial cells significantly down-regulated TNF-α and IL-1β expression of LPS-stimulated PBMC (Figure 1A). In contrast, LPS exposure amplified IL-6 and IL-8 production in mixed co-cultures, compared to PBMC mono-cultures (Figure 1B).
A B
Fig. 1: A549 cells modulate inflammatory response of PBMC alone upon LPS, SEB and PGN stimulation. Mixed co-culture of A549 and PBMC (white bars) or PBMC alone (black bars) were stimulated with 100 ng/ml LPS, 100 ng/ml SEB, 1 µg/ml PGN or PBS (con). After 24 h cell-free supernatants were assayed for TNF-α and IL-1β (A); IL-6 and IL-8 (B) by ELISA. Data are means ± SEM from 11 PBMC donors in 3 independent experiments. TNF-α and IL-1β (C); IL-6 and IL-8 (D) release upon stimulation with PGN and SEB were measured by ELISA. Data are means ± SEM, 8 PBMC donors in 2 independent experiments (Repeated measures ANOVA followed by Bonferroni’s multiple comparison test).
We investigated whether cytokine modulation is similar upon stimulation with other PAMPs. The presence of A549 cells inhibited TNF-α expression also dramatically in PGN- and SEB-activated PBMC. PBMC produced very low IL-1β amounts upon SEB stimulation and cytokine amounts were not affected in co-cultures (Figure 1C). In contrast, PGN activated PBMC to produce IL-1β and its amounts were increased in co-cultures. SEB-stimulated PBMC in mono-culture did not produce IL-6, but a massive IL-6 amplification was present in PGN- and SEB-stimulated co-cultures. We also observed that PGN significantly up-regulated and superantigen tended to increase IL-8 expression in co-culture compared to PBMC alone (Figure 1D).
3.2. Modulation of cytokine secretion by epithelial cells does not require direct cell-cell contact
To find out whether LPS-driven inflammatory effects in the co-culture system are modulated by cell contact mechanisms cells in mono-culture and co-culture were seeded on 24 well normal or transwell plates with 0.4 µm pore size of the membrane inserts (Figure 2). Cytokine release of IL-1β, TNF-α, IL-6 and IL-8 in both settings had similar patterns arguing that the modulation is mediated by soluble factors, not cell-cell contact.
3.3.
αIL-1β, but not αTNF-α neutralizing antibody, inhibits epithelialcell-mediated amplification of IL-6 and IL-8 release in co-culture
It was previously observed that IL-1β and TNF-α induce IL-6 and IL-8 release in A549 cells (Coulter et al., 1999), (Henriquet et al., 2007), (Jiang et al., 1998). We confirmed these results (data not shown) and hypothesized that IL-1β and TNF-α from PBMC mediate the amplification of IL-6 and IL-8 release in LPS-activated co-cultures.
Antibodies against IL-1β and TNF-α were used to neutralize cytokine effects upon LPS stimulation. The neutralizing activity of the αTNF-α and αIL-1β antibodies was demonstrated by inhibited IL-6 and IL-8 production in A549 cells stimulated with TNF-α and IL-1β in quantities released by LPS-stimulated PBMC (data not shown).
Antibodies alone did not induce IL-6 and IL-8 production in non-stimulated cells.
αTNF-α antibody did not affect IL-6 and IL-8 production in LPS-stimulated co-cultures
and PBMC. Addition of αIL-1β to LPS-activated cells resulted in no significant difference in IL-6 and IL-8 amounts between co-cultures and PBMC (Figure 3).
con LPS LPS
Fig. 2: Comparison of cytokine induction in co-culture vs. PBMC upon LPS stimulation in direct cell-cell contact and on transwells. Cells in mixed co-culture (white bars) or PBMC (black bars) were seeded on 24 well plates in direct contact or were physically separated by transwells (polycarbonate membrane, 0.4 µm pore size). After 3 h cells were stimulated with LPS (100 ng/ml) or vehicle (PBS), supernatants were harvested after 24 h and analyzed by ELISA. Data are means ± SEM from 6 PBMC donors in 2 independent experiments (Repeated-measures ANOVA with Bonferroni’s multiple comparison test).
3.4. LPS-induced inhibition of TNF-α in mixed co-culture is regulated on post-transcriptional level
In figure 4 we show that TNF-α down-regulation in LPS-stimulated co-cultures occurs on post-transcriptional level. 3 h LPS stimulation of cells in co-culture or PBMC alone in the presence of 5 µg/ml Brefeldin A blocked extracellular release of TNF-α, as expected. This allowed the observation of a significant down-regulation of intracellular pro-TNF-α in co-culture compared to amounts detected in PBMC.
con
Fig. 3: Effects of αIL-1β and αTNF-α neutralizing antibody on IL-6 and IL-8 production in co-culture vs. PBMC.Cells in co-culture (white bars) or PBMC mono-culture (black bars) were seeded in direct cell-cell contact settings on 48 well plates. In addition, neutralizing TNF-α and IL-1β antibodies (1 µg/ml) were added and after 1 h cells were stimulated with vehicle (con) or 100 ng/ml LPS for 24 h. Supernatants were collected and analyzed for IL-8 and IL-6 expression by ELISA. Data are means ± SEM and represent 2 independent experiments, 8 PBMC donors (Repeated-measures ANOVA with Bonferroni’s multiple comparison test).
3.5. Conditioned supernatant from resting A549 cells induces down-regulation of TNF-α protein and mRNA in LPS-stimulated PBMC
We next tested whether the inhibitory effect on TNF-α release is exerted also by conditioned supernatant from non-stimulated A549 cells. A549 conditioned medium from different time points (2 h, 4 h, 8 h and 24 h) was added to PBMC and after 3 h cells were stimulated with 100 ng/ml LPS for additional 24 h. We observed down-regulated TNF-α secretion by LPS-stimulated PBMC in a time dependent manner
consistent with accumulation of an inhibitory factor in the supernatant of the resting A549 cells (Figure 5).
con Brefeldin A LPS LPS + Brefeldin A 0.0
0.5 1.0 1.5 2.0
2.5
A
- extracellular***
TNF-αααα [ng/ml]
con Brefeldin A LPS LPS + Brefeldin A 0.0
0.2 0.4 0.6
0.8
B
- intracellular***
TNF-αααα [ng/ml]
Fig. 4: Regulation of TNF-α expression upon LPS stimulation occurs on post-transcriptional level, not at release stage. Cells in mixed co-cultures (white bars) vs.
PBMC (black bars) were seeded on 48 well plates and after 3 h were stimulated with vehicle (con) or 100 ng/ml LPS in the presence or absence of 5 µg/ml Brefeldin A. After 3 h supernatants were collected for TNF-α ELISA measurement (A). Cell pellet was resuspended in PBS and 3 freeze-thaw cycles were performed. Cell-free supernatants were harvested and intracellular pro-TNF-α was analyzed by ELISA (B). Data are means ± SEM (8 PBMC donors in 2 independent experiments). Statistical differences are analyzed by repeated-measures ANOVA followed by Bonferroni’s multiple comparison test.
2 4 8 24 con
Fig. 5: Conditioned supernatant from resting A549 cells induces down-regulation of TNF-α in LPS-stimulated PBMC. PBMC were seeded in conditioned supernatant (76%) from A549 (white bars) or medium (black bars) collected after different conditioning times (2h, 4h, 8h and 24h). After 3 h PBMC were stimulated with vehicle (con) or 100 ng/ml LPS for 24 h. Cell-free supernatants were analyzed for TNF-α by ELISA. Data represent mean ± SEM from 3 independent experiments, 9 PBMC donors (Repeated measures ANOVA with Bonferonni’s multiple comparison test).
The fact that the inhibitory factor was produced by resting A549 cells simplified the investigation of whether its effect is already found on mRNA level. Stimulation of PBMC with LPS in the presence of 24 h epithelial-derived supernatant showed a significantly decreased TNF-α mRNA levels (Figure 6).
A549 SN Medium
Fig. 6: Effect of conditioned supernatant from A549 cells on TNF-α mRNA regulation in LPS-activated PBMC. PBMC were seeded in 76% 24 h conditioned supernatant from A549 cells (white bar) or medium (black bar). After 3 h, PBMC were incubated with PBS or stimulated with 100 ng/ml LPS. Expression of TNF-α mRNA was examined by quantitative RT-PCR. Transcript expression was normalized to the housekeeping gene GAPDH. Control values were subtracted from LPS values. Results are means ± SEM from 7 PBMC donors in duplicates in 3 independent experiments (Mann-Whitney Test).
3.6. The TNF-α inhibitory compound appears to be a peptide
We aimed to characterize the TNF-α anti-inflammatory compound in the conditioned supernatant of resting A549 cells. We worked with 76% and 30% supernatants. Our results indicate that TNF-α inhibitory factor is heat and freeze (Figure 7A) stable.
However, 30% conditioned supernatant treatment with 0.5 mg/ml pronase reversed TNF-α levels to those observed in the absence of conditioning. Experiments identifying the size, using a filter with cut-off 10 kDa, have shown that eluted 76%
conditioned medium still possessed strong TNF-α suppressing activity (Figure 7B), similar to observed without filtering and that filtrate of 30% conditioned medium partially lost its activity (figure 7C). These data point towards a peptide as the anti-inflammatory agent.
Fig. 7: TNF-α inhibitory compound appears to be a peptide. A) 30%
conditioned supernatant from A549 cells (A549 30%, white bars) or medium (black bars) was not-treated; heated at 95°C for 15 min; digested with 0.5 mg/ml pronase and 2 x freeze at -80°C. B) 76% (A549 76%) and C) 30% (A549 30%) conditioned supernatant from A549 (white bars) or medium (black bars) was filtrated through a filter with cut-off 10 kDa and eluted fractions were used for experiment. 70µl PBMC were added to 380 µl supernatants and after 3 h were stimulated with vehicle or 100 ng/ml LPS. After 24 h cell-free supernatants were analyzed by TNF-α ELISA. Data are means ± SEM (6 PBMC donors in 2 independent experiments). Repeated measures ANOVA with Bonferroni’s multiple comparison test was applied for statistical differences.
con LPS con LPS con LPS con LPS 0
4. DISCUSSION
Until recently the focus of research into the innate immune response of the lung has been on the role of resident alveolar macrophages. In 2007, a study has shown that various human epithelial cells, including from lung, express functional TLRs, NOD1 and NOD2 to produce anti-microbial products like β-defensin 2 rather than inflammatory mediators (Uehara et al., 2007). In agreement we found that stimulation of A549 cells in mono-culture with LPS, SEB or PGN did not activate these cells to produce IL-1β, TNF-α, IL-6 or IL-8. In addition, Schulz et al. have shown that LPS activates A549 cells to produce IL-6 and IL-8 only in µg doses and in the presence of serum (Schulz et al., 2002). These results indicate that during infection, direct stimulation of airway epithelial cells with bacterial compounds is not a significant source of inflammatory mediators.
To better mimic the in vivo situation in the lung we designed an in vitro co-culture system of A549 cells and PBMC and studied regulation of inflammation upon immune stimulation. Here we show that the presence of lung epithelial cells modulate the immune response of stimulated PBMC. All of the 3 chosen stimuli (LPS, PGN and SEB) induced significantly lower TNF-α, and much higher IL-6 expression in stimulated co-cultures compared to cytokine release in respective stimulated PBMC mono-cultures. Early produced TNF-α cytokine from alveolar macrophages was found to play an important role during initiation and augmentation phases of inflammation (Kelley, 1990), (Strieter et al., 2002). IL-6 was shown as pro-inflammatory regulator of acute phase responses and fever. However, in an LPS-induced acute lung inflammation model IL-6 was described rather as anti-inflammatory cytokine (Xing et al., 1998). Leemans et al. postulated differential roles of IL-6 in lung inflammation by LTA (anti-inflammatory) and PGN (pro-inflammatory effect) both from Staphylococcus aureus (Leemans et al., 2002). These results indicate that indeed epithelial cells may create an anti-inflammatory milieu in the lung through modulation of TNF-α and IL-6 produced from alveolar macrophages.
IL-8 expression in LPS- and PGN-activated co-cultures was also significantly enhanced and tended towards an increase in SEB-stimulated co-cultures. It is known that IL-8 is a very potent chemoatractant for neutrophils in the alveolar space (Coulter et al., 1999), (Reutershan & Ley, 2004), therefore chemokine amounts increase as a prerequisite for resolution of infection.
Modulation of IL-1β expression was stimulus-dependent: lower upon LPS, higher upon PGN and unchanged upon SEB stimulation of co-cultures compared to stimulated PBMC. Although this latter observation was very interesting, regulatory mechanisms for IL-1β production were not further investigated in details.
To study whether LPS-induced inflammatory responses in co-culture require direct cell-cell contact or are driven by soluble mediators, cells were seeded in parallel in mixed and transwell co-cultures. Our results indicated no requirement for cell-cell contact between lung epithelial cells and blood mononuclear cells for all modulated co-culture inflammatory mediators. A critical point of using transwell plates was the choice of the material for membrane inserts. Here polycarbonate material was used, which produced reproducible and reliable results. Polyester membrane inserts from the same company in contrast resulted in confusing and non-reproducible results in comparative experiments. This indicates that the choice of membrane material is crucial and has to be carefully selected according to the application and cell type.
We further investigated soluble mediators involved in LPS-induced mechanisms of IL-6 and IL-8 modulation in co-culture. Interestingly, amplification of LPS-induced IL-6 and IL-8 release has also been reported in A549-neutrophil co-cultures (Grandel et al., 2009). Indeed, stimulation of lung epithelial cells or lung adenocarcinoma cell lines with IL-1β and TNF-α induces expression of IL-6 and IL-8 (Coulter et al., 1999), (Henriquet et al., 2007), (Jiang et al., 1998). A549 cells were also found to express IL-1 type I receptor (Coulter et al., 1999) and TNF-receptor I (Burvall, Palmberg &
Larsson, 2005). Here we show that only neutralizing antibody against IL-1β, but not TNF-α, returns IL-6 and IL-8 expression in LPS-stimulated co-cultures to levels comparable with those in LPS-stimulated PBMC mono-cultures. These results indicate an important role of IL-1β in augmenting the IL-6 and IL-8 response to LPS in the lung. The importance of IL-1β, but not TNF-α, in enhancing IL-8 expression in silica-treated lung cell co-cultures of A549 and THP-1 monocyte-derived cell line was also described (Herseth et al., 2008). One year later, the same group showed that IL-1β is responsible also for increased IL-6 release in silica activated lung cell co-cultures (Herseth et al., 2009).
We further characterized LPS-induced inhibition of TNF-α in co-culture. Brefeldin A blocks protein transport from the endoplasmic reticulum to the Golgi apparatus,
We further characterized LPS-induced inhibition of TNF-α in co-culture. Brefeldin A blocks protein transport from the endoplasmic reticulum to the Golgi apparatus,