American Journal of Respiratory Cell and Molecular Biology

The recruitment of monocytes appears to be a crucial factor for inflammatory lung disease. Alveolar epithelial cells contribute to monocyte influx into the lung, but their impact on monocyte inflammatory capacity is not entirely clear. We thus analyzed the modulation of monocyte oxidative burst by A549 and isolated human alveolar epithelial cells. Epithelial infection with Moraxella catarrhalis induced monocyte adhesion, transepithelial migration, and superoxide generation, whereas stimulation with lipopolysaccharide, tumor necrosis factor-α, interleukin-1β, or interferon-γ induced adhesion or transmigration, but failed to initiate monocyte burst. The effect of microbial challenge was mimicked by phorbol myristate acetate and inhibited by the protein kinase C inhibitor bisindoylmaleimide. Furthermore, evidence for a role of platelet-activating factor–signaling in monocytes is presented. Monocyte burst was neither induced by supernatant nor affected by fixation of A549 cells, excluding the contribution of epithelium-derived soluble factors but emphasizing the mandatory role of intercellular contact. The employment of blocking antibodies, however, denied a role for the adhesion molecules intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, or CD11b/CD18 and CD49d/CD29. In essence, infection of alveolar epithelial cells with M. catarrhalis might amplify the inflammatory capacity of invading monocytes eliciting their superoxide production. The epithelial response to this microbial challenge thus clearly differed from that to proinflammatory cytokines.

CC-chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1 (MCP-1)–driven monocyte accumulation has been implicated in acute and chronic inflammatory lung disease, such as acute respiratory distress syndrome (ARDS) (1), and chronic obstructive pulmonary disease (COPD) (2). In mice, monocytes recruited into the alveolar space in response to CCL2/MCP-1 were “primed” for enhanced responsiveness to lipopolysaccharide (LPS) with increased expression of tumor necrosis factor (TNF)-α (3), and they accelerated LPS-induced neutrophil influx and amplified lung inflammation (4). In patients with ARDS, elevated levels of CCL2/MCP-1 in bronchoalveolar lavage (BAL) fluid as well as distinct and ongoing alveolar monocyte influx were associated with poor oxygenation and worse prognosis (1).

Alveolar epithelial cells (AEpC) cells are a key component of the alveolocapillary barrier. The epithelium protects the host against the outside environment by segregating inhaled foreign agents, and it controls the movement of solutes and water, thereby contributing to the maintenance of lung fluid balance. Type II AEpC synthesize and secrete all components of pulmonary surfactant, making alveolar ventilation and gas exchange feasible at physiologic transpulmonary pressures. Beyond this, AEpC might play a role in pulmonary host defense and inflammation. Secretory products of AEpC were noted to modulate host defense functions of phagocytes. The surfactant proteins A and D enhanced the phagocytosis of certain microorganisms by alveolar macrophages and neutrophils, and surfactant proteins and lipids modulated leukocyte chemotaxis, oxidative burst, and cytokine synthesis (5). AEpC contributed to alveolar monocyte recruitment by the release of CCL2/MCP-1 and upregulation of the adhesion molecules intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 (6). Except for surfactant components, however, it is currently not known if AEpC per se could modulate the inflammatory capacity of invading monocytes.

In addition to their immense capacity producing cytokines, monocytes might trigger inflammatory processes through the production of toxic radicals (7). Because oxidative stress is of great importance in acute (ARDS) and chronic (COPD) lung inflammation, we analyzed the impact of A549 and isolated human type I and type II alveolar epithelial cells on monocyte superoxide production. We investigated the effect of A549 cells stimulated with the proinflammatory cytokines TNF-α, interleukin (IL)-1β, and interferon (IFN)-γ, and we elucidated the impact of AEpC microbial challenge employing the gram-negative organism Moraxella catarrhalis, one of the most important pathogens in lower respiratory tract infections in COPD (8). We ascertained the role of bacterial LPS in this respect, and we analyzed the underlying signaling events. We particularly addressed the role of platelet-activating factor (PAF) for monocyte activation, because PAF is involved in the pathogenesis of lung injury (9), and has been shown to be synthesized by inflammatory AEpC and monocytes (7, 1012).

Monocyte Isolation

Human monocytes were isolated from buffy coats derived from healthy blood donors (kindly provided by the Department of Immunology and Transfusion Medicine, Justus-Liebig-University, Giessen, Germany; approved by the local ethics committee) using a combination of ficoll density gradient centrifugation (800 × g, 30 min, 21°C) and counterflow centrifugal elutriation (Beckmannn JE-5.0 rotor; Beckman Instruments Inc., Palo Alto, CA). Monocyte purity (87–95%) was confirmed by fluorescence-activated cell sorter (FACS) analysis (light scatter characteristics and staining of CD14); cell viability always ranged > 93%. Isolated monocytes were resuspended in serum-free macrophage medium (SFM-M; Invitrogen, Life Technologies, Eggenstein, Germany).

Culture of A549 Cells

A549 cells (CCL 185; American Type Culture Collection, Manassas, VA), a cell line with type II AEpC characteristics, were maintained in HAM's F12 medium containing 10% heat-inactivated fetal calf serum (FCS) and 4 mM L-glutamine (HAM's F12/FCS; Invitrogen) at 37°C and 5% CO2. A quantity of 2 × 104 cells was grown to confluence on 24-well tissue culture plates (Costar, Cambridge, MA). For transepithelial migration experiments, A549 cells were seeded on the lower side of transwell filter inserts (5 μm pore size, 6.4 mm diameter; Costar) to enable leukocyte migration in basal-to-apical direction. When A549 cells reached confluence after 5 d, cells were washed twice before they were forwarded to respective experiments (6).

Isolation and Culture of Human Alveolar Epithelial Cells

Human alveolar epithelial cells (HAEpC) were isolated from human lung tissue obtained from lobectomy specimens distal from tumors as previously described (kindly provided by the Departments of Surgery and Pathology, Justus-Liebig-University; approved by the local ethics committee). Isolated cells were composed of 82–90% epithelial cells (FACS analysis of epithelial cell-specific antigen HEA-125; Camon, Wiesbaden, Germany) and 10–18% leukocytes (light scatter characteristics and expression of CD45). Alkaline phosphatase cytochemistry performed on cytospin preparations revealed 92–97% type II HAEpC in the epithelial cell population (6).

A quantity of 5 × 105 type II HAEpC was seeded on human type IV collagen (Sigma, München, Germany)–coated 24-well tissue culture plates (Costar) or on the lower side of collagen-coated transwell filter inserts (5 μm pore size, 6.4 mm diameter; Costar) and cultured in HAM's F12/FCS containing D-valine (Invitrogen) to prevent growth of fibroblasts. When HAEpC reached confluence after 9 d, cells were washed twice before they were forwarded to respective experiments. At this point of time, cultured HAEpC had lost type II characteristics and evolved the phenotype of type I HAEpC (loss of intracellular alkaline phosphatase, upregulation of ICAM-1, and downregulation of HLA-DR; 6). To analyze HAEpC with type II characteristics, 1 × 106 freshly isolated type II HAEpC were seeded on human type IV collagen–coated 24-well tissue culture plates and incubated in HAM's F12/FCS for 18 h before use.

Bacterial Strain and Growth Conditions

M. catarrhalis (strain 43617, BRO-2 β-lactamase positive; American Type Culture Collection) were grown overnight in brain heart infusion medium (Difco; Becton Dickinson, Heidelberg, Germany) at 37°C in an atmosphere of 5% CO2. Bacterial growth was estimated by measuring the optical density at 600 nm and confirmed by counting cfu after overnight plating. Bacteria were washed twice and resuspended in sterile PBS (Invitrogen). In selected experiments, nonviable organisms were prepared by ultraviolet irradiation under a 15-W ultraviolet lamp for 60 min.

Treatment of A549 and HAEpC

A549 cells were stimulated by the addition of 10 ng/ml TNF-α, 1 ng/ml IL-1β, 100 U/ml IFN-γ, or a combination of 10 ng/ml TNF-α + 1 ng/ml IL-1β + 100 U/ml IFN-γ (R&D Systems, Wiesbaden, Germany) for 4, 12, or 24 h in HAM's F12/FCS, respectively. For microbial challenge, 1 × 105 cfu M. catarrhalis were added to A549, type I, or type II HAEpC monolayers in HAM'sF12/FCS + 1 μg/ml gentamicine (Invitrogen) to prevent excessive growth of bacteria. Plates were centrifuged at 150 × g for 15 min and then incubated for 4, 12, or 24 h at 37°C and 5% CO2. Because M. catarrhalis is a gram-negative organism, the role of endotoxin was examined employing epithelial preincubation with 100 ng/ml purified Salmonella abortus equii endotoxin (Cytogen, Bodenheim, Germany) for 4, 12, or 24 h. In addition, A549 cells were incubated with 1 × 106 cfu ultraviolet-irradiated M. catarrhalis for 24 h.

For transmigration experiments, A549 on the lower surface of filter inserts were immersed in medium containing 10 ng/ml TNF-α and incubated for 4, 12, or 24 h. Alternatively, A549 cells were incubated in medium containing 1 μM phorbol myristate acetate (PMA) for 30 min. For infection experiments, filter inserts were inverted, and the apical side of A549 or type I HAEpC was covered with 50 μl medium containing 1 × 1 05 M. catarrhalis and then incubated for 2 h at 37°C and 5% CO2, followed by immersion in HAM‘s F12/FCS containing gentamicine, and further incubation for 2, 10, or 22 h, respectively.

To elucidate the signal transduction pathway of AEpC activation, microbial challenge of A549 cells was performed in the presence of the protein kinase C (PKC) inhibitor bisindoylmaleimide I (BIM; 0.001, 0.01, 0.1, and 1 μM; Calbiochem, Bad Soden, Germany). In addition, A549 were incubated with 0.001, 0.01, 0.1, 1, or 10 μM of the PKC activator PMA (Sigma). A549 cells were washed vigorously in SFM-M before monocytes were added.

At the end of the respective incubation procedure, monolayer integrity and cell viability were confirmed by light microscopy and lactate dehydrogenase (LDH) measurements (LDH assay; Roche Diagnostics, Mannheim, Germany), respectively. In the time frames tested, monolayer integrity was not disturbed, and the LDH activity in supernatants of A549 or HAEpC incubated with M. catarrhalis, PMA, or BIM remained unchanged compared with that of unstimulated A549 cells (data not shown).

FACS Analysis of Epithelial Adhesion Molecules

FACS analysis of A549 adhesion molecule expression was performed on native or A549 cells activated with 10 ng/ml TNF-α, 1 × 105 M. catarrhalis, or 1 μM PMA. We analyzed the expression of ICAM-1 (MAb R1/1; Bender MedSystems, Vienna, Austria), VCAM-1 (MAb 1G11; Beckman-Coulter, Krefeld, Germany), and P-selectin (CD62P, MAb AC1.2; BD Biosciences Pharmingen, Heidelberg, Germany) by indirect immunofluorescence employing PE-labeled F(ab′)2 fragments of an anti-mouse immunoglobulin antibody (Dianova, Hamburg, Germany), and respective isotype controls.

Synthesis of MCP-1 by A549 Cells

Gene expression of CCL2/MCP-1 in A549 cells stimulated with 1 × 106 M. catarrhalis or 10 ng/ml TNF-α for 8 h, and 10 μM PMA for 30 min was analyzed by RT-PCR. Total cellular RNA was isolated using the acid guanidinium thiocyanate-phenol-chloroform method. The mRNA was reverse transcribed in a GeneAmp PCR System 2400 (Perkin Elmer, Rodgau, Germany), and the polymerase chain reaction (PCR) was performed with first-strand DNA using intron-spanning specific primers for β-actin (5′-AAAGAACCTGTACGCCAACACAGT-GCTGTCT-3′,5′-CGTCATACTCCTGCTTGCTGATCCACATCTG-3′) and MCP-1 (5′-TGAA-GCTCGCACTCTCGCCT-3′, 5′-GTGGAGTGAGTGTTCAAGTC-3′; Stratagene, Heidelberg, Germany). Aliquots of RT-PCR products were electrophoresed through 1.8% (wt/vol) Nusieve/agarose gels and stained with ethidium bromide for ∼ 2 h at 75 V. MCP-1 protein synthesis was quantified by ELISA as described (6). A549 cells were stimulated with 1 × 106 M. catarrhalis or 10 ng/ml TNF-α for 8 h, or 10 μM PMA for 30 min. Thereafter, supernatants were harvested, centrifuged, and stored at −80°C before they were forwarded to ELISA.

Production of Platelet-Activating Factor by A549 Cells and Monocytes

Analysis of PAF production was performed by post-HPLC liquid scintillation counting using the radiochromatogram imaging system (5LS Raytest). PAF production was analyzed in A549 cells infected with 1 × 106 M. catarrhalis for 30 min or 24 h in the presence of 50 μCi [3H]acetate (7.75 Ci/mmol; Amersham, Braunschweig, Germany). Baseline levels were determined in native A549 cells, and activation with 10 μM PMA served as a positive control. PAF production in AEpC-activated monocytes was analyzed in 5 × 107 monocytes cocultured with A549 cells previously infected with M. catarrhalis for 24 h or unstimulated A549 cells. Co-incubation was performed for 30 min in the presence of 50 μCi [3H]acetate before monocytes were removed by vigorous washing. Baseline levels were determined in monocytes co-incubated with unstimulated A549 cells. Reactions were stopped by the addition of three volumes of chloroform:methanol (2:1 vol/vol), and extraction was performed as described (13).

Monocyte Superoxide Production

The oxidative burst of monocytes was analyzed by flow cytometry as previously described (14). This assay is based on the oxidation of the nonfluorescent dye hydroethidine (HE; Molecular Probes, Göttingen, Germany) to the fluorescent dye ethidium bromide in the presence of superoxide radicals. Elutriated monocytes were loaded with 1 μM HE in SFM-M for 10 min at 37°C, washed twice, and resuspended in SFM-M.

HE-labeled monocytes (1 × 105) were incubated alone, or they were added to native or activated A549 cells or type I and type II HAEpC in 24-well plates for 30 min. For transepithelial migration experiments, 1 × 106 monocytes were added to the upper compartment of transwell chambers containing native or stimulated A549 cells or type I HAEpC on the lower side of the filter inserts. Monocytes were allowed to transmigrate the epithelial barrier for 120 min at 37°C in the basal-to-apical direction. The incubation was stopped by the addition of ice-cold PBS containing 5 mM EDTA (Sigma). Wells were incubated for further 5 min on ice, rinsed vigorously, and detached monocytes were transferred into vials containing ice-cold PBS + 5mM EDTA + 1% paraformaldehyde. Fixed monocytes were immediately forwarded to flow cytometric measurement.

Monocyte Adhesion and Transepithelial Migration

Isolated monocytes were radiolabeled with 5 μCi 111Indium (10 mCi/ml In111-chloride; Amersham) tropolone (Fluka, Neu-Ulm, Germany) as previously described (6). Labeled monocytes were suspended in SFM-M, and 5 × 105 monocytes were added to each well of native or stimulated A549 cells. After co-incubation for 30 min at 37°C, nonadherent monocytes were removed by three gentle washing steps with warm SFM-M, and adherent monocytes were lysed by the addition of 0.5% Triton-X 100 (Sigma). Lysed monocytes were removed by three washing steps. The fractions of both nonadherent and adherent cells were counted in a γ counter, and for each sample the fraction of adherent monocytes (%) was calculated from: counts of adherent monocytes/(counts of nonadherent + adherent monocytes).

For transepithelial migration experiments, 1 × 106 radiolabeled monocytes were added to the upper compartment of transwell chambers containing native or stimulated A549 cells on the lower side of the filter units. Monocytes were allowed to transmigrate the epithelial barrier for 120 min at 37°C in the basal-to-apical direction. The monocytes in the lower compartment were lysed by the addition of 0.5% Triton X-100, and lysed cells were counted in a γ-counter to determine the number of migrated cells. The number of monocytes which migrated through the epithelial barrier was expressed as percentage of counts in the lower compartment in relation to counts initially added to the upper compartment.

Flow Cytometric Analysis

Flow cytometric measurements were performed on a FACScan from Becton Dickinson (Heidelberg, Germany). Data were acquired and analyzed using the CellQuest research software (Becton Dickinson). A549 cells and monocytes were gated by forward and right angle light scatter properties, and PE- as well as HE-derived fluorescence was analyzed using a 570/26-nm bandpass filter. Expression of surface molecules is given as mean fluorescence intensitiy (MFI), and activated superoxide production is calculated as Δ MFI = MFI of HE-labeled activated monocytes − MFI of HE-labeled, unstimulated monocytes incubated in SFM-M alone.

Experimental Protocols

For the induction of monocyte burst in the absence of AEpC, 1 × 105 HE-labeled monocytes were mock-incubated or incubated for 30 min with 0.001, 0.01, 0.1, 1, or 10 μM PMA, or with 1 × 106 M. catarrhalis in SFM-M.

To analyze the induction of monocyte adhesion, transepithelial migration or superoxide production by AEpC, 111In- or HE-labeled monocytes were added to unstimulated or previously activated A549 cells or HAEpC for 30 min (adhesion) or 120 min (transmigration), respectively. In oxidative burst experiments, HE-labeled monocytes incubated for 30 or 120 min in SFM-M alone served as baseline control.

To elucidate the role of epithelium-derived soluble factors versus contact-dependent mechanisms in the activation process of monocyte burst, unstimulated, PMA-activated, or Moraxella-infected A549 cells in the lower compartment were co-incubated with HE-labeled monocytes in the upper compartment of transwell chambers for 30 min, employing filter inserts with a pore diameter of 0.4 μm (Costar) to prevent intercellular contact. In addition, PMA-stimulated or Moraxella-infected A549 cells in 24-well plates were fixed with 1% paraformaldehyde for 10 min at room temperature, followed by several vigorous washing steps before the addition of monocytes.

The contribution of monocyte adhesion molecules to adhesion and activation was studied by the use of adhesion blocking mAbs against CD11b (clone 44, R&D), CD18 (clone IB4; generated from mouse hybridoma HB-10164, ATCC), CD49d (clone HP2/1, anti–α4-chain of the VLA-4 integrin; Serotec, Oxford, UK), and CD29 (clone 4B4, anti-β1 chain of the VLA-4 integrin; Beckmann-Coulter). Monocytes were loaded with HE or 111In, respectively, and subsequently incubated with saturating amounts of blocking mAbs or isotype controls for 30 min at room temperature, before they were added to A549 cells.

The contribution of epithelial adhesion molecules to monocyte adhesion and activation was studied employing the above-mentioned adhesion blocking mAbs against ICAM-1 and VCAM-1. Unstimulated cells or A549 activated with 10 ng/ml TNF-α and 1 × 105 M. catarrhalis for 24 h, or 1 μM PMA for 30 min were incubated with saturating amounts of mAbs or isotype controls for 60 min at 37°C before the addition of monocytes.

The impact of epithelial-derived CCL2/MCP-1 on monocyte adhesion and activation was examined using a neutralizing anti-human CCL2/MCP-1 mAb (R&D; 15). Native or activated A549 cells were preincubated with anti-CCL2/MCP-1 for 30 min before monocytes were added.

The contribution of PAF to the process of monocyte adhesion and activation was analyzed by the use of the PAF antagonists BN52021 (Ginkolide B; Biomol, Hamburg, Germany), BN50730 (a generous gift from Dr. P. Braquet, Institute Henri Beaufour, Le Plessis Robinson, France), or CV-6209 (Biomol, Hamburg, Germany). In the absence or presence of 0.001, 0.01, 0.1, 1, or 10 μM BN, or 10 μM CV-6209 HE- or 111In-labeled monocytes were co-incubated with A549 cells prestimulated with 1 μm PMA for 30 min, or 1 × 105 M. catarrhalis for 24 h, respectively. In addition, HE-labeled monocytes were incubated in absence or presence of 0.001, 0.01, 0.1, or 1 μM PAF (Calbiochem) for 30 min in Hanks' balanced salt solution (HBSS; Invitrogen) in the absence of AEpC.

Statistical Analysis

Data were expressed as mean ± SEM. For analyzing statistical difference, nonparametric Kruskal-Wallis test followed by Dunns post test were performed. Statistically significant differences were defined as P values less than 0.05.

Infection of AEpC with M. catarrhalis Induced Adhesion, Transepithelial Migration, and Superoxide Production in Monocytes

Infection of A549 cells with 1 × 105 M. catarrhalis time-dependently induced monocyte adhesion (Figure 1A)

and transepithelial migration (Figure 1B), and simultaneously provoked monocyte superoxide production (Figure 1C). In almost the same manner as A549 cells, M. catarrhalis–infected primary human type I HAEpC also induced superoxide production in adhering and transmigrating monocytes (Figure 1D). Likewise, freshly isolated type II HAEpC evoked a lower but still significant oxidative burst response in monocytes (type II HAEpC + 1 × 105 M. catarrhalis (24 h): 72 ± 14 ΔMFI, mock-infected type II HAEpC: 13 ± 8 ΔMFI; each compared with baseline control, mean ± SEM, n = 3). The induction of monocyte burst depended on the infection with viable bacteria, because incubation of A549 cells with ultraviolet-irradiated M. catarrhalis failed to initiate superoxide generation (Figure 1C). Approving the stimulatory effect of A549 cells and HAEpC, co-incubation of monocytes with 1 × 106 M. catarrhalis in SFM-M failed to evoke a significant burst reaction (Figure 1C). Not until fresh human serum containing complement was added did monocytes co-incubated with M. catarrhalis produce oxygen radicals, most likely by the induction of phagocytosis. This human serum did not contain significant amounts of LPS as was tested by Limulus assay (< 10 pg/ml), and serum heated to 56°C for 30 min did not evoke a burst response in monocytes co-incubated with M. catarrhalis (data not shown).

AEpC Stimulated with Proinflammatory Cytokines Induced Monocyte Adhesion and Transepithelial Migration but Failed to Initiate Superoxide Production

In contrast to M. catarrhalis–infected A549 cells, stimulation of A549 cells with TNF-α increased monocyte adhesion (Figure 2A)

and transepithelial migration (Figure 2B), but failed to initiate a monocyte burst reaction (Figure 2C). Likewise, activation of A549 cells with IL-1β and IFN-γ, or the combination of TNFα + IL-1β + IFN-γ, did not initiate oxygen radical production in adherent monocytes (Figure 2D). In contrast to M. catarrhalis, incubation of A549 cells with LPS did not provoke superoxide generation in added monocytes (Figure 2D).

The Activation of Epithelial PKC Is Required for the Induction of Superoxide Generation in Co-Cultured Monocytes

Infection of A549 cells with M. catarrhalis in the presence of the PKC inhibitor BIM dose-dependently abrogated the stimulatory effect on monocyte burst (Figure 3A)

. Accordingly, preactivation of A549 cells with the PKC activator PMA initiated superoxide production in monocytes (Figure 3B) and increased monocyte adhesion to the epithelium (Figure 3C). Interestingly, PMA stimulation of A549 cells did not increase monocyte transepithelial migration (monocyte migration across native A549 monolayers: 9.8 ± 2.3%, versus 6.1 ± 2.7% migration across PMA-stimulated A549; data are given as mean ± SEM, n = 5 each). Incubation of monocytes with PMA in the absence of epithelial cells only slightly increased their superoxide production, again approving the stimulatory capacity of AEpC (Figure 3B).

The Activation of Monoycte Burst by AEpC Depended on Intercellular Contact

Co-culture of monocytes with PMA-activated or M. catarrhalis-infected A549 cells in transwell chambers under nonadherent conditions did not provoke superoxide generation (Figure 4B)

. Paraformaldehyde-fixed PMA-activated or M. catarrhalis–infected AEpC continued to provoke superoxide production in added monocytes (Figure 4A).

AEpC-Derived CCL2/MCP-1 Promoted Monocyte Adhesion but Did Not Contribute to the Activation Process of Monocyte Burst

M. catarrhalis upregulated gene expression as well as CCL2/MCP-1 protein synthesis in A549 cells within 8 h of co-incubation (Figure 5)

. In contrast, PMA failed to increase CCL2/MCP-1 expression within 30 min (Figure 5; which sufficed to initiate A549-induced monocyte activation [Figure 3]), whereas TNF-α upregulated CCL2/MCP-1 synthesis (Figure 5), but failed to initiate monocyte burst (Figure 2). These findings argued against a role of epithelial CCL2/MCP-1 in monocyte activation, and employing a neutralizing mAb against CCL2/MCP-1, we could show that CCL2/MCP-1 contributed to monocyte adhesion induced by TNF-α or M. catarrhalis (Figure 6A), but that it was not involved in the A549-induced activation process of monocyte burst (Figure 6B).

Neither the Monocyte Adhesion Molecules CD11b/CD18 and CD49d/CD29, nor the Epithelial Adhesion Molecules ICAM-1 and VCAM-1, Were Involved in the A549-Induced Activation Process of Monocytes

Because monocyte adhesion to A549 cells was essentially required for the induction of oxidative burst (Figure 4), we examined whether monocyte or epithelial adhesion molecules contributed to this event. At first, we analyzed the impact of microbial challenge, PMA activation, or TNF-α stimulation on the expression of A549 adhesion molecules (Table 1)

TABLE 1. Facs analysis of adhesion molecule expression on A549 cells

A549 (MFI)

Isotype Control
Native120 ± 10350 ± 24200 ± 18112 ± 25
10 ng/ml TNF-α115 ± 18690 ± 87 (P < 0.01)388 ± 32 (P < 0.01)129 ± 19
1 μM PMA131 ± 21370 ± 56246 ± 49135 ± 17
1 × 105 M. catarrhalis
139 ± 17
511 ± 41 (P < 0.01)
342 ± 28 (P < 0.01)
133 ± 8

Definition of abbreviations: FACS, fluorescence-activated cell sorter; ICAM-1, intercellular adhesion molecule-1; PMA, phorbol myristate acetate; TNF-α, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1.

Native cells or A549 stimulated with TNF-α and M. catarrhalis for 24 h, or PMA for 30 min, were incubated with saturating amounts of isotype controls or specific mAbs against ICAM-1, VCAM-1, or P-Selectin. Data are presented as mean fluorescence intensity (MFI; mean ± SEM, n = 5 each; P values compare respective results with corresponding MFI of native A549).

. A549 cells expressed significant amounts of ICAM-1 and low levels of VCAM-1 under baseline conditions, and activation with TNF-α for 24 h markedly upregulated ICAM-1 and significantly increased the expression of VCAM-1. Likewise, incubation with M. catarrhalis for 24 h induced upregulation of ICAM-1 and VCAM-1, whereas incubation with PMA for 30 min did not alter the expression profile of these molecules. Because P-selectin, which is typically induced on endothelial cells and platelets, was noted to induce superoxide production in monocytes (16), we additionally analyzed the expression of P-selectin on A549 cells. But neither TNF-α, nor PMA and M. catarrhalis induced the expression of P-selectin on A549 cells (Table 1), and FACS analysis of elutriated monocytes ruled out that monocyte-adherent platelets might account for monocyte activation (analysis of P-selectin and GPIIb/IIIa expression on monocytes; data not shown).

Considering the baseline expression of A549 adhesion molecules and activation of monocyte integrins by extracellular matrix proteins, we performed blocking experiments employing neutralizing mAbs. Blocking epithelial ICAM-1 or VCAM-1, and monocyte CD11b/CD18 or CD49d/CD29 significantly decreased monocyte adhesion to TNFα stimulated A549 cells (Figure 6A), and inhibition of ICAM-1 or CD11b/CD18 slightly reduced monocyte adhesion to M. catarrhalis-infected A549 cells. In contrast, neither anti-ICAM-1 and anti-VCAM-1, nor mAbs against CD11b/CD18 and CD49d/CD29 inhibited adhesion to PMA-activated A549 cells (Figure 6A). Blocking mAbs against ICAM-1 and VCAM-1, or CD11b/CD18 and CD49d/CD29 did not inhibit the oxidative burst response in monocytes co-cultured with PMA-activated A549 cells, and they also failed to attenuate superoxide production induced by M. catarrhalis-infected A549 cells (Figure 6B).

PAF Antagonists Decreased Superoxide Production in Monocytes and Reduced Adhesion to M. catarrhalis–Infected or PMA-Activated AEpC

Monocyte superoxide production was significantly reduced when co-incubation with PMA-activated or M. catarrhalis–infected A549 cells was performed in the presence of the natural Ginkolide PAF receptor antagonist BN52021, or the synthetic compound BN50730 (Figure 7A)

. Moreover, both antagonists inhibited monocyte adhesion to PMA- or Moraxella-activated A549 cells (Figure 7B). Likewise, 10 μM of the PAF receptor antagonist CV-6209 inhibited monocyte superoxide production induced by PMA-activated A549 cells (monocytes + native A549 cells: 6 ± 5 ΔMFI, monocytes + A549 prestimulated with 1 μM PMA: 138 ± 17 ΔMFI, monocytes + native A549 + 10 μM CV-6209: 9 ± 4 ΔMFI, monocytes + PMA [1 μM]-stimulated A549 + 10 μM CV-6209: 83 ± 10 ΔMFI; each compared with baseline control, mean ± SEM, n = 3). PAF antagonists did not affect the viability of monocytes or A549 cells, respectively (data not presented).

Although we detected PAF production in A549 cells after stimulation with PMA, microbial challenge with M. catarrhalis did not induce PAF synthesis in A549 cells, whether short-term incubated with AEpC for 30 min, or long-term incubated for 24 h (Figure 7C). However, analysis of monocytes previously added to M. catarrhalis–infected A549 cells for 30 min revealed a significant increase of PAF synthesis compared with monocytes co-incubated with mock-infected A549 cells (Figure 7C). In the absence of A549 cells, exogenous PAF evoked a slight but not significant burst signal in monocytes (Figure 7D).

The recruitment of neutrophils is generally considered to play a central role in the development of lung injury, but the contribution of monocytes to lung inflammation and tissue injury is less well defined. Recent work from our group showed that elevated BAL levels of the monocyte-specific chemokine CCL2/MCP-1, and a distinct and ongoing accumulation of monocytes within the alveolar space, correlated with poor oxygenation and worse prognosis in patients with ARDS (1). Likewise, the intratracheal administration of CCL2/MCP-1 with consecutive alveolar monocyte accumulation amplified the LPS-induced pulmonary inflammatory response in mice, enhancing the impairment of alveolocapillary barrier function and increasing protein leakage (4). These findings implicated a detrimental role of monocyte recruitment for lung inflammation. In patients with COPD, pulmonary inflammation is associated with increased numbers of neutrophils in the airways, along with a marked expansion of the macrophage population. Increased BAL levels of CCL2/MCP-1 in these patients also implicated enhanced monocyte recruitment, presumably promoting pulmonary inflammation and lung tissue damage by the release of cytokines and toxic radicals (2).

Type I HAEpC and type II AEpC were noted to contribute to inflammatory monocyte accumulation, secreting CCL2/MCP-1 in response to TNF-α (6). The present study demonstrates that not only TNF-α, but also microbial challenge with M. catarrhalis, provoked CCL2/MCP-1 synthesis and upregulated the adhesion molecules ICAM-1 and VCAM-1 in A549 cells, and subsequently induced monocyte adhesion and transepithelial migration. Beyond attracting monocytes, M. catarrhalis–infected A549 cells, but not cytokine-activated cells, exerted critical impact on monocyte inflammatory capacity, inducing a strong respiratory burst response in adherent or transmigrating cells. The response profile of A549 cells to microbial challenge with M. catarrhalis thus clearly differed from that to inflammatory stimulation with TNF-α, IL-1β, or IFN-γ, implicating differential regulation of AEpC-induced leukocyte adhesion, migration and activation. The suppression of the A549 stimulatory capacity by PKC inhibition and its mimicry by PMA stimulation suggested a crucial role of epithelial PKC for this event.

The promotion of host defense functions of transmigrating leukocytes has been previously documented for intestinal epithelial cells. Intestinal epithelium augmented the phagocytic capacity of transmigrating neutrophils, but, in contrast to the present study, they did not upregulate neutrophil oxidative burst (17). This discrepancy may be attributed to the migration process across nonactivated intestinal epithelial cells in that study, encouraging our hypothesis that the initiation of superoxide production in transmigrating monocytes may specifically rely on microbial challenge with M. catarrhalis and activation of epithelial PKC. In the absence of AEpC, monocytes produced only minor amounts of superoxide in response to PMA, and M. catarrhalis failed to provoke a burst response under serum-free culture conditions. These results clearly approved the direct stimulatory capacity of activated AEpC.

M. catarrhalis has emerged as an important cause of lower respiratory tract infections, particularly in patients with COPD (8). Our data presented evidence for a direct inflammatory activation of AEpC by this organism. Beyond provoking monocyte-specific chemotactic activity, we noted a differential induction of inflammatory cytokines in A549 cells as M. catarrhalis induced the expression of IL-8, IL-6, and GM-CSF, but failed to upregulate IL-1β, RANTES (Regulated on Activation, Normal T Expressed and Secreted), or inducble nitric oxide synthase (our own, unpublished data). Activation of airway epithelium by M. catarrhalis may thus suffice to initiate an inflammatory host defense response in the lower respiratory tract. Apart from phagocytosing leukocytes, this direct stimulation of host cells may not be restricted to AEpC, because M. catarrhalis has recently been shown to activate mast cells and B lymphocytes (18, 19). Host cell activation might be induced by cell wall components of M. catarrhalis. Notwithstanding the fact that M. catarrhalis is a gram-negative organism expressing lipooligosaccharides (8), and A549 cells as well as HAEpC express the LPS receptor TLR-4 (20), endotoxin was not responsible for the inflammatory activation of A549 cells. Alternatively, Moraxella peptidoglycan (21) may stimulate AEpC via TLR-2 (22), or the outer membrane proteins (OMP) of M. catarrhalis (8) may account for inflammatory activation, as has recently been demonstrated for OMP derived from Klebsiella pneumoniae (23). However, nonviable M. catarrhalis did not provoke epithelial activation in our experiments, which argued against a role for these molecules. However, it has to be kept in mind that damage of these structures by ultraviolet irradiation, or insufficient production of peptidoglycan up to the time of bacterial killing has not been ruled out in this study. Moreover, intracellular organisms like Chlamydia pneumoniae (24), or exotoxins derived from extracellular bacteria (25) are known to induce inflammatory activation of respiratory epithelial cells. Because there is no evidence for the production of exotoxins by M. catarrhalis (8), our data thus implicated host cell invasion by M. catarrhalis as the underlying event of AEpC activation. In fact, confocal microscopy revealed invasion of M. catarrhalis into A549 cells and BEAS2b, a SV-40 transformed human bronchial epithelial cell line (H. Slevogt and J. Seybold, personal communication).

Our data suggested that AEpC or monocyte surface molecules, rather than soluble factors secreted by the activated epithelium, induced oxygen radical production in monocytes. The AEpC adhesion molecule ICAM-1 has previously been shown to support pulmonary host defense against Klebsiella pneumoniae, augmenting the phagocytic capacity of adherent alveolar macrophages (26). Although unstimulated A549 cells expressed abundant levels of ICAM-1, which was even upregulated after infection with M. catarrhalis, ICAM-1 as well as VCAM-1 were not involved in the activation process of adherent monocytes in the present study. In contrast, mAbs against both, ICAM-1 and VCAM-1, reduced monocyte adhesion to M. catarrhalis–infected A549 cells and markedly inhibited adhesion to TNF-α–activated A549 cells, therefore demonstrating their blocking efficiency.

The monocyte ligands of ICAM-1 and VCAM-1, CD11b/CD18 and CD49d/CD29, respectively, are also known to interact with extracellular matrix proteins produced by AEpC (6). In addition, extracellular matrix proteins were noted to amplify the oxidative burst capacity of monocytes (27), and monocyte activation via β1- and β2-integrins is a well known phenomenon (28, 29). Thus, one might argue that microbial challenge or PMA stimulation might have disturbed the monolayer integrity of HAEpC or A549 cells, thereby initiating monocyte superoxide production by adhesion to newly exposed matrix proteins. However, upregulation of monocyte superoxide production by matrix proteins required at least 18 h of co-incubation (27), and transepithelial migration of monocytes across TNF-α–stimulated A549 cells also allowed interaction with epithelial matrix proteins, but did not provoke monocyte superoxide production. Furthermore, mAbs against CD11b and CD18, or CD49d and CD29, did not inhibit the induction of monocyte burst, although mAbs against CD11b and CD18 slightly reduced monocyte adhesion to M. catarrhalis–infected A549 cells, and mAbs against both, β1- and β2-integrins significantly inhibited adhesion to TNF-α–stimulated A549 cells. It is therefore unlikely that contact of monocytes with matrix proteins induced monocyte superoxide production. Collectively, these results further support the differential regulation of AEpC-induced leukocyte adhesion and activation, and they implicate the existence of adhesive interactions between monocytes and AEpC beyond the “classical” adhesion molecule pathways.

A549 cells infected with M. catarrhalis synthesized CCL2/MCP-1, and the recombinant protein has been shown to induce chemotaxis, calcium flux, and respiratory burst in monocytes (30). In addition, surface-bound chemokines co-located with adhesion molecules were known to activate adherent leukocytes (31), and CCL2/MCP-1 may act on monocytes in concert with P-selectin (16). However, stimulated A549 cells did not express P-selectin, and a neutralizing mAb against CCL2/MCP-1 failed to inhibit monocyte activation. Nevertheless, A549-derived CCL2/MCP-1 promoted monocyte adhesion to A549 cells infected with M. catarrhalis or stimulated with TNF-α, and CCL2/MCP-1–driven monocyte adhesion has been previously described for human pulmonary artery endothelial cells (15). Indeed, these findings argued against a role of CCL2/MCP-1 for monocyte activation. AEpC-derived CCL2/MCP-1 may thus primarily account for the recruitment of monocytes rather than enhancing their inflammatory capacity. This is in agreement with our observation that short-time incubation of A549 cells with PMA induced monocyte adhesion and respiratory burst, but alongside with missing upregulation of CCL2/MCP-1, ICAM-1, and VCAM-1, it did not provoke transepithelial migration, further supporting the concept of the differential regulation of leukocyte recruitment and activation by AEpC.

A549 cells have been previously shown to produce PAF in response to PMA (10), and PAF was noted to induce superoxide production in macrophages (32, 33) and monocytes (7). Although we confirmed PAF synthesis in PMA-activated A549 cells, and PAF receptor antagonists diminished A549-induced monocyte burst, we were not able to detect PAF production in A549 cells infected with M. catarrhalis. Furthermore, exogenous PAF did not induce significant superoxide production, hence suggesting the induction of endogenous PAF synthesis in monocytes by the activated epithelium. Similarly, the PAF receptor antagonist WEB2179 reportedly inhibited the PMA-induced oxidative burst in rat Kupfer cells, whereas exogenous PAF did not suffice to induce superoxide production (34). This finding also implicated autocrine PAF synthesis mediating subsequent superoxide generation in these cells. The PAF receptor antagonist BN50730 has the ability to cross cell membranes (35), and in our experiments it exhibited a more pronounced effect than BN52021 or CV-6209, which mostly operated extracellularly. These results also supported our hypothesis of autocrine PAF production in activated monocytes, and indeed, we were able to demonstrate upregulation of PAF synthesis in monocytes after 30 min of co-incubation with previously infected A549 cells. In essence, these data implicated that activated AEpC induced monocyte superoxide production via autocrine synthesis of PAF; however, PAF initiated the burst reaction not until monocytes were primed by intercellular contact with the activated epithelium. This priming phenomenon has also been observed in peritoneal macrophages (32); likewise, intracellular but not exogenous PAF has been shown to regulate eicosanoid generation in these cells (36). Activation of monocytes via CD11b/CD18 or monocyte binding to P-selectin have been described to initiate their PAF production (11, 12), but contribution of these molecules was excluded in our experiments. Although monocyte adhesion to inflammatory A549 cells occurred in part by yet undefined mechanisms, we also showed that PAF contributed to this process. Since PAF is involved in the pathogenesis of lung injury (9), and monocyte recruitment appeared to be detrimental in the course of pulmonary inflammation, one might speculate that AEpC-induced, and PAF-mediated monocyte superoxide production may contribute to this event under certain conditions.

In conclusion, the present study lends further credence to the concept that AEpC play an important role in alveolar host defense mechanisms and pulmonary inflammation. Moreover, differential regulatory events are suggested to induce different types of epithelial activation. One type is provoked by proinflammatory cytokines and is characterized by epithelial monocyte adhesion and transmigration in the absence of significant monocyte respiratory burst, and largely depends on “classical” adhesion molecule interactions. Another type is provoked by microbial challenge with M. catarrhalis, and similarly evokes adhesion and transmigration of monocytes, but is accompanied by a marked respiratory burst as the most prominent feature. Signaling events in this latter type of response are suggested to include epithelial PKC activation and autocrine PAF signaling in monocytes, with no major contribution of the “classical” adhesion molecule mechanisms. These findings clearly demonstrate that AEpC may not only induce alveolar leukocyte recruitment by the release of chemokines and the expression of adhesion molecules, but they may also regulate the state of leukocyte activation dependent on the type of epithelial stimulation. However, the present study did not explore the role of surfactant in the process of monocyte activation evoked by M. catarrhalis–infected AEpC. As surfactant components produced by type II AEpC have been shown to be important regulators of leukocyte host defense functions, their impact on AEpC-mediated monocyte activation deserves further elucidation.

The excellent technical assistance of M. Muhly and M. Lohmeyer is greatly appreciated.

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Correspondence and requests for reprints should be addressed to Simone Rosseau, M.D., Department of Internal Medicine and Infectious Diseases, Charité–Campus Mitte, Schumannstrasse 20/21, 10117 Berlin, Germany. E-mail:


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