Role of alveolar epithelial ICAM-1 in lipopolysaccharide-induced lung inflammation

Material

Except where noted, all reagents were purchased from Sigma (Buchs, Switzerland).

Animals

Pathogen-free male Long-Evans rats (250–300 g) were purchased from M & B, Ry, Denmark. Rats were anaesthetized with subcutaneously administered Hypnorm® (fentanyl/fluonisone; Janssen, Beerse, Belgium) 0.25 mL·kg body weight⁻¹, Domitor® (medetomidine; Pfizer, Inc., Westchester, PA, USA) 0.25 mL·kg body weight⁻¹ and 0.1% atropine 0.05 mL·kg body weight⁻¹. All animal experiments and animal care were approved by the Swiss Veterinary Health Authorities.

Isolation and culture of alveolar epithelial cells

AECs from rat lungs were collected following previously described protocols 11, 12. Cells were grown to confluence over a 3-day period. Purity was assessed using a fluorescein-labelled lectin, Griffonia simplicifolia I (macrophage staining).

Stimulation with lipopolysaccharide and cytokines

Confluent AECs were stimulated overnight with various agents: LPS (Escherichia coli, serotype 055:B5) at 100 µg·mL⁻¹, recombinant mouse tumour necrosis factor-α (TNF-α) at 20 ng·mL⁻¹ and interferon gamma (IFN-γ) at 500 U·mL⁻¹. Prior to cell stimulation, the medium was changed to Dulbecco modified Eagle medium (DMEM)/1% foetal bovine serum (FBS).

Northern blot for intercellular adhesion molecule-1

Ribonucleic acid (RNA) was extracted from confluent monolayers of unstimulated (control) and stimulated AECs using TRIZOL® (Life Technologies, Basel, Switzerland), chloroform (Fluka, Buchs, Switzerland) and isopropanol (Fluka). The RNA (5 µg) was loaded on to a 1% agarose/formaldehyde gel. After electrophoresis, the RNA was transferred to a nylon Zeta-Probe® blotting membrane (BioRad, Hercules, CA, USA). Equal loading was confirmed by rehybridization with β-actin. The RNA was cross-linked using an ultraviolet filter and the blots were prehybridized for 30 min. Hybridization was performed overnight with a ³²P-labelled probe using a random priming method (Life Technologies). The blots were exposed to XAR5 Kodak film at −70°C.

Enzyme-linked immunosorbent assay for intercellular adhesion molecule-1 expression

To determine ICAM-1 expression on the surface of AECs, a cell-based enzyme-linked immunosorbent assay (ELISA) was performed according to a previous protocol 13. Final optical density (OD) at 492 nm was calculated by subtracting the OD of cells incubated with the secondary antibody alone or with a control antibody (mineral oil-induced plasmocytoma cell (MOPC) 21, mouse immunoglobulin-G (IgG)) (Pharmingen, San Diego, CA, USA) from the OD of cells incubated with primary and secondary antibody together.

Preparation of neutrophils

Whole blood was obtained from healthy human volunteers and neutrophils were isolated as described previously 13, 14. Neutrophils were pre-incubated with blocking antibodies to FcRII (mouse antihuman CD32) and FcRIII (mouse antihuman CD16, Pharmingen) (10 µg·mL⁻¹) at 37°C for 15 min, washed and added to the wells.

Assay of neutrophil adherence to alveolar epithelial cells

AECs were added to 96-well plates at a density of 5×10⁴ cells·well⁻¹ and grown to confluence at 37°C in 5% carbon dioxide. Cells were stimulated with 100 µg·mL⁻¹ LPS overnight, washed six times and then incubated with monoclonal mouse anti-ICAM-1 (10 µg·mL⁻¹, 1A29; Serotec, Oxford, UK) or control antibody (10 µg·mL⁻¹, MOPC-21) at 37°C in 5% CO₂ for 20 min. After washing the cells once with DMEM/10% FBS, 1×10⁵ neutrophils, previously incubated with FcRII and FcRIII, were added to each well and incubated at 37°C in 5% CO₂ for 30 min. Nonadherent cells were removed by washing each well four times with culture medium. The remaining neutrophils were counted immediately using a cell chamber.

Alveolar macrophages: isolation, preparation and adherence to alveolar epithelial cells

Rat alveolar macrophages were harvested as described previously 15. Unstimulated and stimulated AECs were incubated with anti-ICAM-1 (1A29) or control antibody (MOPC-21) at a concentration of 10 µg·mL⁻¹ at 37°C for 20 min. Macrophages were preincubated with blocking antibodies to FcRII and FcRIII, as described above. AECs were washed and incubated with macrophages at 37°C in 5% CO₂ for 30 min. Nonadherent cells were washed away and adherent cells were counted.

Cytotoxicity assay

Cytotoxicity was measured using a standard lactate dehydrogenase (LDH) assay (Promega, Madison, WI, USA). AECs were seeded into the wells of a 96-well culture dish, as described above. Cells were stimulated with 100 µg·mL⁻¹ LPS overnight. Neutrophils (1×10⁶) in phosphate-buffered saline (PBS; pH 7.4)/0.02% bovine serum albumin (BSA) were added to each well. The neutrophils were allowed to settle on to the epithelial cell monolayer for 30 min before being stimulated with phorbol 12-myristate 13-acetate (PMA) (100 ng·well⁻¹). After incubation for 2 h, the supernatant was removed from each well and centrifuged at 700×g for 5 min. Some AECs were incubated with lysing solution (from assay kit) for 45 min (target cell maximum LDH release). Supernatant was also collected from wells of AECs that had not been incubated with neutrophils (spontaneous LDH release). Cytotoxicity was calculated according to the following formula: where ER is experimental release, SR is spontaneous release, and TR is total release.

Fluorescence staining for intercellular adhesion molecule-1 in lipopolysaccharide-injured lungs

LPS resuspended in PBS or PBS alone (control animals) was applied intratracheally, as described previously 16. After 6 h, rat lungs were frozen in liquid nitrogen. Dual staining was performed with anti-ICAM-1 and a monoclonal goat antimouse vascular endothelial (VE) cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Sections were incubated with control antibody or with the secondary antibody alone. All antibodies were diluted in PBS/1% BSA. Slides were fixed with 3% paraformaldehyde (PFA), blocked with PBS/10% FBS and incubated overnight with anti-VE cadherin at a concentration of 10 µg·mL⁻¹ in PBS/1% BSA/0.3% Triton X-100 at 4°C. Incubation with a flurorescein isothiocyante (FITC)-labelled secondary rabbit antigoat IgG followed. Slides were then fixed with 3% formalin, which was followed by ICAM-1 staining. Mouse antirat ICAM-1 was diluted to 1 µg·mL⁻¹ and incubated with the slides at 4°C for 1 h. Staining with a secondary Cy3-goat antimouse IgG conjugate (1:500, Jackson Immunoresearch Laboratories, Inc., West Grove, PA, USA) was performed. Finally, sections were covered with mounting medium (DAKO Corporation, Carpinteria, CA, USA) containing 1,4-diazabicyclo[2.2.2]octane. Immunofluorescence staining was examined using a confocal laser scanning microscope (Zeiss, Jena, Germany).

A second dual staining was performed with anti-ICAM-1 and a type II cell antibody directed against the type IIb sodium phosphate (NaPi) cotransporter, which is exclusively found in the apical membrane of type II cells of the alveolar epithelium 17. Sections were first incubated with anti-ICAM-1 and the secondary Cy3-labelled antibody as described above. Sections were then fixed with 3% PFA and incubated overnight with anti-NaPi-IIb antiserum (diluted 1:500 in PBS/1% BSA/0.3% Triton X-100) at 4 °C. A secondary FITC-labelled pig antirabbit antibody (DAKO Corporation) was added. The final steps were the same as described above.

In vivo blocking studies with intercellular adhesion molecule-1 antibody

Rats were anaesthetized as described above. LPS (150 µg), together with 200 µg polyclonal rabbit antirat ICAM-1, was instilled intratracheally in 300 µL PBS 18. IgG was used as a control. Bronchoalveolar lavage (BAL) was performed and cells were counted and stained with Diff Quick (Dade Behring, Duedingen, Switzerland) after 4 h of injury. Lungs were homogenized and myeloperoxidase (MPO) levels were determined after 240 s, as previously described 19.

Lactate dehydrogenase release

In order to evaluate the toxicity of the cytokine and LPS concentrations employed, LDH levels in the supernatants of stimulated and unstimulated cells were measured using a nonradioactive assay system (Promega).

Statistical analysis

All experiments were performed at least three times. Three different sets of ELISAs were analysed. Analysis of variance (ANOVA) with post-ANOVA comparison was performed to assess the significance of differences. A p-value of <0.05 was considered significant. All values are expressed as mean±sem.

Characterization of alveolar epithelial cells

Monolayers of AECs were stained to detect macrophages. There was <5% contamination. If the cultures were <95% pure, they were not used for further experiments.

Upregulation of rat intercellular adhesion molecule-1 and its messenger ribonucleic acid in stimulated alveolar epithelial cells

In order to define the expression pattern of ICAM-1 under different stimulation conditions, AECs were exposed overnight to several agonists, including LPS (100 µg·mL⁻¹), recombinant mouse TNF-α (20 ng·mL⁻¹) and recombinant rat IFN-γ (500 U·mL⁻¹). LDH assays demonstrated all the concentrations described above to be nontoxic (data not presented). The relatively high concentration of LPS was chosen in accordance with the in vivo assay and pre-existing data 13. In addition, concentration-dependent ICAM-1 expression revealed the same results with 10 and 100 µg·mL⁻¹ LPS stimulation. Figure 1a⇓ shows upregulation of ICAM-1 in AEC primary culture 24 h after stimulation with any of the three agonists. LPS enhanced ICAM-1 expression by 39%, TNF-α by 21% and IFN-γ by 13%. ICAM-1 expression on stimulated cells was significantly increased compared to expression on control cells (p<0.01). At the messenger RNA (mRNA) level there was a similar result, with the most prominent upregulation of ICAM-1 occurring under LPS stimulation, as assessed by Northern blot (fig. 1b⇓).

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Fig. 1.—

Upregulation of a) rat intercellular adhesion molecule-1 (ICAM-1) protein and b) ICAM-1 messenger ribonucleic acid (mRNA) in alveolar epithelial cells. Cells were unstimulated (control (C)) or stimulated with lipopolysaccharide (LPS; 100 µg·mL⁻¹), recombinant mouse tumour necrosis factor-α (TNF-α; 20 ng·mL⁻¹) or recombinant rat interferon-γ (IFN-γ; 500 U·mL⁻¹) for 24 h. a) ICAM-1 measured by enzyme-linked immunosorbent assay (ELISA). Three ELISAs were evaluated. Data are expressed as mean±sem. b, c) Northern blot (5 µg ribonucleic acid). Equal loading was shown by rehybridization with β-actin (c). All stimulated cells showed increased ICAM-1 mRNA levels, with LPS being the most potent agent. **: p<0.01 versus control.

Time course of upregulation of rat intercellular adhesion molecule-1 and its messenger ribonucleic acid in stimulated alveolar epithelial cells

A time course was established for upregulation of ICAM-1 and ICAM-1 mRNA in primary cultures of AECs stimulated with LPS. Cells were incubated for 2, 4, 6, 8 and 24 h with LPS (100 µg·mL⁻¹) and a cell-based ELISA for ICAM-1 was performed. Control cells were exposed to PBS instead of LPS. Maximal expression of ICAM-1 occurred after 6 h (36% increase; p<0.01) (fig. 2a⇓). Upregulation of ICAM-1 mRNA is shown in figure 2b⇓. Early expression started after 2 h with sustained expression for ≤24 h. A relatively high baseline expression of ICAM-1 was found. This finding is in contrast with the result with L2 cells, where there was only slight expression in unstimulated cells 13.

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Fig. 2.—

Time course of upregulation of a) intercellular adhesion molecule-1 (ICAM-1) protein and b) ICAM-1 messenger ribonucleic acid (mRNA) in alveolar epithelial cells incubated with LPS. a) ICAM-1 measured by enzyme-linked immunosorbent assay (ELISA). Three ELISAs were evaluated. A 36% increase in ICAM-1 was seen after 6 h. Data are expressed as mean±sem. b, c) Northern blot (5 µg ribonucleic acid). Equal loading was shown by rehybridization with β-actin (c). Maximal expression of ICAM-1 mRNA occurred within 2 h. **: p<0.01 versus time 0 (control).

As only a 40% increase in cell surface ICAM-1 expression was seen after LPS stimulation, compared to the significant increase in ICAM-1 mRNA levels, soluble ICAM-1 concentrations in the media were measured by Western blot. Interestingly, there was no difference between ICAM-1 concentrations in the supernatant from control cells and LPS-stimulated cells.

Adherence of neutrophils and alveolar macrophages to alveolar epithelial cells

In order to evaluate the biological implications of ICAM-1 expression on AECs, adherence assays were performed with neutrophils and alveolar macrophages. Neutrophil adherence to unstimulated AECs was appreciable (35 adherent cells·square⁻¹) (fig. 3⇓). Compared to unstimulated AECs, the adherence of neutrophils to LPS-stimulated AECs increased by 114% (75 adherent cells·square⁻¹). Neutrophil adherence to stimulated AECs decreased by 40% (p<0.001) in the presence of blocking antibodies to ICAM-1. At the beginning of the experiment, different antibody concentrations in the range 5–100 µg·mL⁻¹ were evaluated, with 10 µg·mL⁻¹ being the most efficient concentration concerning blockade.

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Fig. 3.—

Adherence of neutrophils to alveolar epithelial cells (AECs) unstimulated (control; □) or stimulated (└) overnight with lipopolysaccharide (LPS; 100 µg·mL⁻¹). Blocking intercellular adhesion molecule-1 (ICAM-1) with monoclonal mouse antirat ICAM-1 (1A29; 10 µg·mL⁻¹) resulted in decreased adherence of neutrophils to AECs compared to control antibody (40% less on stimulated cells). The results of three single assays were analysed. Data are expressed as mean±sem. **: p<0.001.

Similar adherence assays were performed with rat alveolar macrophages obtained from normal rat lungs by BAL. Adherence to nonstimulated AECs was 38 cells·square⁻¹, increasing to 55 cells·square⁻¹ in LPS-stimulated AECs (45% increase). Blocking of ICAM-1 on the surface of AECs before incubation with macrophages resulted in a decrease in adhesion to stimulated AECs by 28% (p<0.001) (fig. 4⇓).

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Fig. 4.—

Adherence of alveolar macrophages to alveolar epithelial cells (AECs) unstimulated (control; □) or stimulated (└) overnight with lipopolysaccharide (LPS; 100 µg·mL⁻¹). A modest decrease in adherence of macrophages to stimulated AECs (28%) occurred in the presence of anti-intercellular adhesion molecule-1 (ICAM-1). The results of three single assays were analysed. Data are expressed as mean±sem. **: p<0.001.

Damage to alveolar epithelial cells caused by neutrophils

The ability of neutrophils to damage AECs was examined. PMA-stimulated neutrophils were incubated with LPS-stimulated AECs or with control cells for 2 h. Figure 5⇓ shows LDH release as an indirect measure of cell death. Neutrophils as a source of possible LDH release could be excluded by trypan blue staining, which showed them to be intact. Control AECs exposed to stimulated neutrophils showed a cytotoxicity of 30%, whereas that of LPS-stimulated AECs increased to 54% (p<0.005).

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Fig. 5.—

Alveolar epithelial cell (AEC) killing by phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils (100 ng·well⁻¹). AECs were unstimulated (control) or stimulated overnight with lipopolysaccharide (LPS; 100 µg·mL⁻¹) and then incubated with PMA-stimulated neutrophils for 2 h. Data are expressed as mean±sem. The experiment was carried out three times with similar results. p<0.005.

In vivo upregulation of rat intercellular adhesion molecule-1 in lungs after stimulation with lipopolysaccharide

In order to study upregulation of airway ICAM-1 by LPS in vivo, sections of LPS- or PBS-treated lungs were used for immunostaining to assess ICAM-1 content. Costaining with VE cadherin and ICAM-1 showed clear ICAM-1 expression on endothelial cells in control lungs (fig. 6a⇓). The pattern in LPS-stimulated lungs was similar, but with a clear increase in nonendothelially localized ICAM-1 (fig. 6b⇓).

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Fig. 6.—

Immunofluorescence staining of intercellular adhesion molecule-1 (ICAM-1) in rat lung after harvesting 6 h after intratracheal instillation of: a, c) phosphate-buffered saline (PBS); and b, d) lipopolysaccharide (LPS). Frozen sections were immunostained for ICAM-1 (red) and vascular endothelial (VE)-cadherin (green; a and b) or the type IIb sodium phosphate cotransporter (green; c and d). a) The yellow/orange colour represents double staining for ICAM-1 and VE-cadherin (arrow). The asterisk indicates a vessel lumen with yellow/orange vascular endothelial lining. b) The red lined area (arrow) shows epithelial ICAM-1. Internal scale bars=a, b) 20 µm and c, d) 10 µm.

In addition, type II cell staining was performed to evaluate ICAM-1 expression on type I and type II cells. Interestingly, there was no ICAM-1 expression on type II cells in control animals (fig. 6c⇑) or LPS-stimulated animals (fig. 6d⇑).

Airway blocking of intercellular adhesion molecule-1

Airway ICAM-1 was blocked using a polyclonal antibody 18. Of the BAL fluid cells analysed with Diff Quick, 95% were neutrophils and 5% macrophages. After ICAM-1 blockade, the total BAL fluid cell content was reduced by 50% (p<0.05) (fig. 7⇓), whereas MPO levels were reduced by 35% (p<0.01) compared to controls (fig. 8⇓).

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Fig. 7.—

Total cell count in bronchoalveolar lavage fluid from rat lung after intratracheal intercellular adhesion molecule-1 (ICAM-1) blockade in the lipopolysaccharide (LPS)-injured lung. Lungs were instilled with phosphate-buffered saline (PBS; □) or LPS (└) in the presence of immunoglobulin-G (IgG) or anti-ICAM-1 (200 µg·mL⁻¹). The vascular system of the lungs was flushed 4 h later and the bronchoalveolar system lavaged four times with cold PBS. Blocking airway ICAM-1 reduced the cell count by 50% in bronchoalveolar lavage fluid. Data are expressed as mean±sem (n=5). *: p<0.05.

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Fig. 8.—

Neutrophil content of rat lung after intratracheal intercellular adhesion molecule-1 (ICAM-1) blockade in the lipopolysaccharide (LPS)-injured lung. Lungs were instilled with phosphate-buffered saline (PBS; □) or LPS (└) in the presence of immunoglobulin-G (IgG) or anti-ICAM-1 (200 µg·mL⁻¹). The vascular system of the lungs was flushed 4 h later and the bronchoalveolar system lavaged. This was followed by determination of myeloperoxidase (MPO) levels, a measure of neutrophil content. Blocking airway ICAM-1 reduced MPO levels by 35%. Data are expressed as mean±sem (n=5). AU: arbitrary units. **: p<0.01.