Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Airway inflammation and the bronchial hyperresponsiveness it triggers are hallmarks of asthma (1, 2). A key feature of the inflammatory response seen in asthma is eosinophil accumulation in the airways (3, 4). Little is known, however, about the mediators that trigger eosinophil-epithelial adhesion or the pathways that facilitate eosinophil retention in the airways. In contrast, those governing eosinophil-endothelial interactions and the initial steps of eosinophil migration from the blood vessel have been extensively investigated (5). Eosinophil cell adhesion molecules (CAM) that have been identified to date include (1) the ₁ integrin, very late antigen-4 (VLA-4; ₄₁); (2) the ₂ integrins, lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) and macrophage antigen-1 (Mac-1; CD11b/ CD18); (3) L-selectin, P-selectin glycoprotein ligand-1 (PSGL-1), and an E-selectin ligand (ESL)-bearing sialyl-Lewis X; and (4) ₄₇ (8, 9). CAM identified on primary cultures of airway epithelial cells are intercellular adhesion molecule-1 (ICAM-1), CD44, and LFA-3 (10). In contrast to endothelial cells, P- or E-selectin, which bind PSGL-1 or ESL-1, respectively (9), were not detected on airway epithelial cells and there is conflicting evidence as to whether vascular cell adhesion molecule-1 (VCAM-1), a ligand for VLA-4 and ₄₇ (8), is expressed (11). Thus, of the CAM known to date, interactions of the CD18 integrins with ICAM-1 would be the most likely to mediate eosinophil adhesion to airway epithelium.

ICAM-1, a member of the immunoglobulin gene superfamily (15), is expressed constitutively at very low levels, or not at all, on primary airway epithelial cells or cell lines (10, 11, 16). The role of ICAM-1 in airway inflammation is therefore thought to result from a distinct pattern of induction following exposure to interferon (IFN-) and, to a lesser extent, tumor necrosis factor- (TNF-) or interleukin-1 (IL-1; 10, 11, 13, 16). Increased concentrations of IL-1, TNF-, and IFN- in asthmatic bronchoalveolar lavage (BAL) fluid and/or culture supernatant from BAL leukocytes have been measured (17). IFN- has also been shown to enhance TNF-- or IL-1-induced ICAM-1 expression on airway epithelial cells (11) and, in this way, may trigger the increased epithelial ICAM-1 detected in bronchial mucosa biopsies from asthmatics (20, 21).

Alteration of eosinophil CAM expression may also facilitate eosinophil accumulation in asthmatic airways. In support of this, sputum eosinophils, compared with eosinophils in blood, show increased expression of CD11b, suggesting that upregulation of this CAM has occurred in the passage from blood to airway (22). The inflammatory mediators C5a, FMLP, platelet-activating factor (PAF), the C-C chemokines eotaxin and RANTES (regulated on activation, normal T cell expressed and secreted) as well as the phorbol ester phorbol myristate acetate (PMA), have been shown to increase expression and/or activation of CD11b/ CD18 on eosinophils to varying degrees (23). The role of PAF and leukotriene (LT) B₄, another eosinophil-active mediator, has been extensively studied in the pathogenesis of asthma (28). There is also evidence for the involvement of C5a (29, 30) and the contribution to asthma of the C-C chemokines has recently begun to emerge (31, 32).

Further support for a role in asthma of ICAM-1 and the CD18 integrins comes from in vivo studies that show blocking antibodies against CD18 and/or ICAM-1-inhibited airway eosinophilia in animal models of asthma (33- 35). It is not clear from these studies, however, whether anti-CAM antibodies act, in part, at the level of eosinophil-epithelial adhesion and, if they do, whether CD18 interaction with ICAM-1 is involved. In vitro studies that investigated neutrophil-epithelial adhesion have suggested that a non-ICAM-1 ligand for CD18 may be expressed on human airway epithelial cells (10, 36). Adhesion studies with PMA- or IL-5-stimulated eosinophils have led to the suggestion that eosinophils may also adhere to airway epithelium in a CD18-dependent/ICAM-1-independent manner (13, 39). In contrast, respiratory syncytial virus (RSV)- infected type II alveolar epithelial cells (A549) have been shown to support PMA-stimulated eosinophil adhesion via a CD18/ICAM-1-dependent pathway (40). Thus, the role of ICAM-1 as a ligand for CD18 in eosinophil-epithelial interactions induced by inflammatory mediators, likely to be present in allergic airways, is not fully understood. The aims of the present study therefore were (1) to assess the effect(s) on eosinophil adhesion to normal human bronchial epithelial cells (NHBEC) of the eosinophil- active inflammatory mediators C5a, FMLP, LTB₄, PAF, eotaxin, RANTES, and macrophage inflammatory protein-1 (MIP-1), in comparison with PMA; and (2) to investigate, using blocking antibodies, the role(s) of CD18 and ICAM-1 in eosinophil-NHBEC adhesion.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Culture Reagents

NHBEC or human lung microvascular endothelial cells (HLMVEC), prepared by Clonetics (San Diego, CA), were purchased as cryopreserved first passage (1°) or third passage (3°) cultures, respectively, from TCS Biologicals Ltd. (Buckingham, UK). NHBEC from three different donors were used for this study. Bronchial epithelial cell growth medium (BEGM), microvascular endothelial growth medium (EGM-MV), 0.025% trypsin + 0.01% EDTA, Hanks' balanced salt solution (HBSS), and trypsin neutralizing solution were also obtained from TCS Biologicals Ltd. A549 cells (type II alveolar epithelial cell carcinoma) were obtained from ATCC (Rockville, MD). Dulbecco's modified Eagle's medium (DMEM; high glucose), heat-inactivated fetal calf serum (FCS), glutamine, penicillin, streptomycin, Dulbecco's phosphate-buffered saline (PBS) (± Ca²⁺/Mg²⁺), were purchased from GIBCO Laboratories (Paisley, Scotland).

Cytokines and Other Reagents

Human recombinant (hr) TNF- was obtained from Boehringer Mannheim UK (Lewes, East Sussex, UK; specific activity > 1 × 10⁸ U/mg, respectively). Human recombinant IFN- was obtained from R&D Systems (Abingdon, Oxfordshire, UK; specific activity 1 × 10⁷ U/mg). Human eotaxin, RANTES, and MIP-1 were obtained from Peprotech EC Ltd. (London, UK). C5a was a gift from Dr. J. J. van Oostrum (Ciba-Geigy, Summit, NJ). LTB₄ was obtained from Cascade (Reading, Berkshire, UK) and C-16 PAF from Bachem Ltd. (Saffron Walden, Essex, UK). The following products were purchased from Sigma Chemical Company Ltd. (Poole, Dorset, UK): PMA, FMLP, 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), gelatin, goat serum, horse serum, hydrogen peroxide (H₂O₂), dimethyl sulfoxide (DMSO), potassium chloride (KCl), calcium chloride (CaCl₂), and magnesium sulphate (MgSO₄). Percoll and dextran were obtained from Pharmacia Biotech Ltd. (St. Albans, Hertfordshire, UK), sterile normal saline (0.9%) from FL (Manufacturing) Ltd., Fresenius Health Care Group (Basingstoke, Hampshire, UK) and very low endotoxin bovine serum albumin (BSA) from Bayer Ltd. (Basingstoke, UK). Citric acid, EDTA, di-sodium hydrogen orthophosphate (Na₂HPO₄), sodium dihydrogen orthophosphate (NaH₂PO₄) and D-glucose were obtained from BDH Ltd. (Poole, Dorset, UK). MACS CS separation columns and CD16 microbeads were purchased from Miltenyi Biotech (Bisley, Surrey, UK). Calcein-AM was obtained from Cambridge Bioscience (Cambridge, UK).

Antibodies

Affinity-isolated goat antimouse peroxidase conjugate gamma and light chain specific, was obtained from TCS Biologicals Ltd. Mouse antihuman ICAM-1 (RR1/1) IgG₁ whole mAb (41), for use in ELISA, was provided by Dr. R. Rothlein (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT). For use in adhesion assays, F(ab')₂ fragments of a mouse antihuman ICAM-1 (BBIG-I1) IgG₁ whole mAb (42), provided by Dr. R. Pigott (British Biotech, Oxford, UK), were generated by Cymbus Biotechnology Ltds. (Chandlers Ford, Hampshire, UK). Mouse IgG_1-k (MOPC21, whole mAb and F(ab')₂ fragments), and mouse antihuman CD18 (6.5E) IgG₁ whole mAb were gifts from Dr. M. Robinson (Celltech, Slough, Berkshire, UK); mouse antihuman IgG₁ whole mAb against VLA-4 (2B4) (43), and VCAM-1 (BBIG-V1) were from Dr. R. Pigott.

Cell Culture

The NHBECs used in this study were characterized by morphological appearance through serial passage and were maintained in BEGM, an epithelial selective medium that is a modification of LHC-9 medium, supplemented with 52 µg/ml bovine pituitary extract, 0.5 ng/ml hr epidermal growth factor (EGF), 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 10 µg/ml transferrin, 0.5 µg/ml epinephrine, 6.5 ng/ ml triiodothyronine, 0.1 ng/ml retinoic acid, 50 µg/ml gentamicin, and 50 ng/ml amphotericin-B. NHBECs, purchased from Clonetics, have previously been used as an in vitro model of bronchial epithelium with which to investigate eosinophil-epithelial adhesion (13). Confluent cells were washed with HBSS, trypsinized using 0.025% trypsin + 0.01% EDTA, and collected into trypsin neutralizing solution. Cells were seeded at a density of 3.2 × 10³ cells per well onto 1% gelatin-coated flat-bottomed Nunclon 96-well microtiter plates. Five days after seeding, confluent monolayers were used for ELISA or adhesion assays. The confluent cell density was approximately 1 × 10⁴ cells per well and this was consistent between replicate wells and plates.

HLMVECs, also purchased from Clonetics, were prepared from minced peripheral lung lobes and have been shown to retain a number of properties of endothelial cells (EC), including the production of human F-VIII-related antigen, uptake of acetylated low-density lipoprotein and expression of CD31 (44). Cells were maintained in EGM-MV medium, a modification of MCDB 131, supplemented with 10 ng/ml hrEGF, 1 µg/ml hydrocortisone, 5% heat-inactivated FCS, 50 µg/ml gentamicin, 50 ng/ml amphotericin-B, and bovine brain extract containing 12 µg/ml protein and 10 µg/ml heparin. HLMVECs were subcultured as for NHBEC and were used in adhesion assays 4 d after seeding; confluent cell density was approximately 8.5 × 10³ cells/well.

A549 were maintained in DMEM containing 10% heat-inactivated FCS, 4 mM glutamine, 200 U/ml penicillin, and 200 µg/ml streptomycin. Confluent cells were trypsinized (with 0.05% trypsin + 0.02% EDTA) and seeded at a confluent cell density of 4 × 10⁴ cells per well onto 96-well plates. Monolayers were used in adhesion assays 2 d after seeding.

ELISA for Cell Adhesion Molecule Expression

ICAM-1 was detected by an ELISA method (45) using a mouse antihuman ICAM-1 (RR1/1) primary mAb and a peroxidase-linked goat antimouse secondary antibody. Briefly, confluent NHBEC monolayers in Nunclon 96-well plates were incubated with TNF- (1 ng/ml), IFN- (10 ng/ml) or TNF- in combination with IFN- for 6, 24, or 72 h. Cytokines were diluted in serum-supplemented complete culture media. Following removal of stimuli, cells were washed three times with PBS containing Ca²⁺, Mg²⁺, and 0.1% BSA and incubated for 45 min with 1 µg/ml RR1/1 or mouse myeloma protein (MOPC21) diluted in complete medium. Primary antibody was removed by washing and NHBEC monolayers were incubated (45 min) with a 1:1,000 dilution (in PBS + 10% goat serum) of goat antimouse peroxidase conjugate followed by incubation with a peroxidase-sensitive substrate, ABTS (1 mg/ml, in 0.2 M citrate/phosphate buffer, pH 5, containing 0.1% H₂O₂), for 30 min. The reaction was terminated by addition of 0.2 M citrate. All incubations were carried out at room temperature. Chromophore development was determined by measuring optical density (OD) at 405 nm (OD₄₀₅) using a Titretec MCC/340 Multiscan microplate reader (Flow Laboratories, Hertfordshire, UK). Background absorbance was determined from monolayers incubated without primary antibody and this value was then subtracted from the absorbance readings. Reported data are derived from OD readings that fall along the linear portion of the development curve. Adhesion molecule expression is given as OD. In experiments to determine whether VCAM-1 was expressed on NHBEC, a mouse antihuman VCAM-1 (4B2) primary mAb was substituted for RR1/1 in the ELISA detailed previously.

Separation of Human Peripheral Blood Eosinophils or Neutrophils

Granulocytes were isolated from peripheral blood of normal or mildly atopic adult donors for neutrophils or eosinophils, respectively, by the method of Haslett and colleagues (46 as described by Burke-Gaffney and Hellewell [47]). Briefly, blood was collected to a total volume of 40 ml into 3.8% citrate and spun for 20 min at 300 g. The platelet-rich plasma was removed, underlaid with 90% Percoll, and spun at 2,000 g for 20 min at room temperature (temperature used unless otherwise stated) to produce platelet-poor plasma (PPP). To the lower buffy coat produced by the first spin, 5 ml of 6% dextran was added and the volume was made up to 50 ml with 0.9% saline. The mixture was allowed to stand for 30 min for erythrocyte sedimentation to occur. The leukocyte-rich supernatant was removed and centrifuged at 300 g for 8 min. The pellet was resuspended in PPP and layered onto freshly prepared discontinuous Percoll-plasma gradients (42% and 51% Percoll in PPP) and centrifuged for 10 min at 260 g. The granulocyte band was collected, washed in PPP, and resuspended in Krebs Ringer phosphate dextrose (KRPD) buffer (4.8 mM KCL; 3.1 mM NaH₂PO₄; 12.5 Na₂HPO₄:5% vol/vol glucose; 2% vol/vol FCS). Granulocytes obtained from normal donors were > 98% neutrophils. Granulocytes obtained from mildly atopic donors were used to purify eosinophils and were incubated with anti-CD16 microbeads to remove neutrophils (1/10 dilution when 40 µl of beads were added per 10⁸ granulocytes for 45 min). A Type-CS magnet-activated cell separation column was set up in a magnetic separator and microbead-labeled granulocytes applied to the top of the column. Eosinophils (> 97% pure) were eluted in 35 ml KRPD buffer. Eosinophils or neutrophils (5 × 10⁶ cells/ml) were labeled (30 min, 37°C) with a fluorescent dye, calcein-AM (10 µM dissolved in 1% DMSO in KRPD without FCS). Cells were washed twice in KRPD without FCS and resuspended at 1 × 10⁶ cells/ml in KRPD containing 2.5% FCS, 0.93 mM CaCl₂, and 1.2 mM MgSO₄ for the adhesion assay.

Measurement of Eosinophil or Neutrophil Adhesion to Epithelial or Endothelial Monolayers

NHBEC monolayers grown on 96-well plates were incubated with complete culture medium or TNF- (1 ng/ml) and IFN- (10 ng/ml) in combination for 24 h. Monolayers were washed three times with PBS (containing Ca²⁺/Mg²⁺) to remove stimuli before carrying out the adhesion assay. To set up adhesion assays, 100 µl of KRPD with Ca²⁺/Mg²⁺ or two times the final concentration of mAbs or stimuli were added per well in experiments to investigate the effects of stimuli or mAb alone. Alternatively, 50 µl each of four times the final concentration mAb and stimuli were added to investigate effects of blocking mAb on stimulated eosinophils. Final concentrations of mAb and stimuli are given in the RESULTS section. One hundred microliters of calcein-AM-labeled granulocytes were then added to the assay plate and incubated at 37°C for 7.5, 15, 30, 60, 90, or 120 min as indicated in the results. Fluorescence was measured using a Biolite F1 plate reader (excitation wavelength of 485 ± 25 nm and emission wavelength of 530 ± 25 nm) before and after washing the plate to remove nonadherent cells by two gentle washes with PBS containing 1% horse serum (48). Results were expressed as percent adhesion calculated from the fluorescence measured after washing the plate minus the background fluorescence, divided by the fluorescence before washing plate minus background times 100, unless otherwise stated.

To assess whether eosinophils had migrated through the NHBEC monolayers during the adhesion assay, wells were washed vigorously four times with PBS (without Ca²⁺/Mg²⁺) to remove adherent eosinophils without damaging the monolayers and the fluorescence measured. Less than 5% of total fluorescence added was associated with the monolayers at 2 h, and at 30 min the fluorescence was similar to the background for control monolayers incubated without eosinophils. These data suggest that little or no eosinophil migration occurred in our studies.

Statistics

Results are expressed as mean ± SEM of n experiments, unless otherwise stated. Statistical analysis was carried out using a one-way analysis of variance followed by the Student-Newman-Keuls multiple comparison test, which compares all values with one another unless otherwise stated. Instat GraphPad software was used to perform statistical analysis. Results were deemed significant if P < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

TNF-/IFN- Increases ICAM-1 Expression on NHBEC

A low level of constitutive ICAM-1 expression was detected on resting NHBEC monolayers, as the OD₄₀₅ measured after incubation of monolayers with anti-ICAM-1 mAb (0.27 ± 0.01, n = 8) was significantly greater (P < 0.001) than that measured when monolayers were incubated with a control antibody, MOPC21 (0.19 ± 0.01, n = 8), or without primary mAb (background OD₄₀₅; 0.18 ± 0.01, n = 8); ICAM-1 expression after background subtraction was therefore 0.09 ± 0.02 (n = 8).

ICAM-1 expression was significantly increased at 6 h following incubation with IFN- (10 ng/ml) or TNF- (1 ng/ml) + IFN- (Figure 1a). At 24 h, the TNF- + IFN--induced ICAM-1 expression was significantly greater (P < 0.001) than the IFN--induced expression (Figure 1b), and the IFN-- or TNF- + IFN--induced ICAM-1 expression remained elevated at 72 h (Figure 1c). TNF- alone did not significantly increase ICAM-1 expression at 6, 24, or 72 h. In contrast to ICAM-1, VCAM-1 expression was not detected on resting or cytokine-activated NHBEC under the conditions used in this study (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 1.   ELISA determination of ICAM-1 expression on NHBEC following exposure to culture medium, TNF- (1 ng/ml), IFN- (10 ng/ml), or TNF- + IFN- for (a) 6, (b) 24, or (c) 72 h. Results are shown as means ± SEM of three to six experiments. *P < 0.05, **P < 0.01, or ***P < 0.001, respectively, denote significant ICAM-1 induction compared with treatment with culture medium. #P < 0.001 denotes a significant difference compared with OD405 measured following IFN- treatment for 24 h.

Basal Eosinophil Adhesion to Resting NHBEC Increases with Time and Is CD18 Independent

Eosinophil adhesion to NHBEC increased with time (up to 120 min as shown in this study; Figure 2) and Table 1 shows a significant increase (P < 0.05) in eosinophil adhesion measured at 60 min, compared with 30 min, for six different donors. Anti-CD18 mAb (6.5E; 20 µg/ml) did not alter adhesion of human eosinophils to resting NHBEC monolayers measured at 7.5, 15, 30, 60, 90, or 120 min compared with a control antibody, MOPC21 (20 µg/ ml) or KRPD buffer alone (Figure 2). Also, a mAb against VLA-4 (2B4; 20 µg/ml), alone or in combination with 6.5E, and an anti-ICAM-1 mAb (BBIG-I1; 10 µg/ml), had no effect on basal adhesion (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 2.   Kinetics of eosinophil adhesion to unactivated NHBEC and effects of anti-CD18 mAb (6.5E). Adhesion was measured at 7.5, 15, 30, 60, 90, and 120 min in the presence of KRPD buffer or 20 µg/ml of MOPC21 or 6.5E. Results were expressed as means ± SEM of three separate experiments.

To establish whether the increase in eosinophil adhesion with time was specific for NHBEC, we investigated adhesion to A549 alveolar epithelial cells and HLMVEC. Adhesion to A549 was significantly greater (P < 0.05) at 60 min than 30 min, whereas adhesion to HLMVEC was not significantly different (Table 1). Time-dependent, CD18-independent basal adhesion to NHBEC was not specific for eosinophils as neutrophil adhesion also increased with time (data not shown); neutrophil adhesion, in the presence of 20 µg/ml 6.5E (19.7 ± 2.8% at 120 min), was not reduced compared with control (20.9 ± 1.7%, n = 3).

TNF-/IFN- Activation of NHBEC Triggers a CD18/ ICAM-1-dependent Increase in Eosinophil Adhesion

Eosinophil adhesion to TNF-/IFN--activated NHBEC also increased with time (up to 120 min in this study; Figure 3a). Adhesion measured at 90 or 120 min, but not at 7.5, 15, 30, or 60 min, was significantly greater (P < 0.05 or 0.01, respectively) than eosinophil adhesion to resting NHBEC measured at the corresponding time (Figure 3a). Eosinophil adhesion to cytokine-activated NHBEC at 120 min was also measured in the presence of 6.5E, 2B4, or MOPC21 (20 µg/ml) and F(ab')₂ fragments of BBIG-I1 or MOPC21 (10 µg/ml). Basal eosinophil adhesion to resting NHBEC (normalized to 100%) was increased to cytokine-activated NHBEC in the presence of MOPC21 (Figure 3b). 6.5E significantly reduced adhesion (P < 0.05) to basal levels, whereas 2B4 had no significant effect (Figure 3b). BBIG-I1 also significantly (P < 0.001) reduced adhesion to basal levels (Figure 3c). A small, but significant (P < 0.05), increase in neutrophil adhesion to activated NHBEC (23 ± 3%) compared with resting NHBEC (16.5 ± 0.7%, n = 4), was also measured (120 min) and 6.5E or BBIG-I1 inhibited this increased adhesion (data not shown).

View larger version (14K): [in this window] [in a new window]   Figure 3.   Kinetics of eosinophil adhesion to cytokine-activated NHBEC and effects of mAb against CD18 (6.5E), VLA-4 (2B4), or ICAM-1 (BBIG-I1). NHBEC were pretreated for 24 h with culture medium (open circle) or TNF- (1 ng/ml) + IFN- (10 ng/ml; filled circle or bar). Eosinophil adhesion to resting or activated NHBEC was measured at (a) 7.5, 15, 30, 60, 90, or 120 min; (b) at 120 min in the presence of KRPD buffer or 20 µg/ml of MOPC21 control antibody, 6.5E, or 2B4; and (c) at 120 min in the presence of KRPD buffer or 10 µg/ml of F(ab')2 fragments of control antibody, MOPC21, or BBIG-I1. Results are means ± SEM of three to five experiments (a and b) or six replicates from two experiments (c) and are expressed as percent adhesion (a) or percent increase (b and c) of basal adhesion at 120 min where this is normalized to 100%. *P < 0.05, **P < 0.01, and ***P < 0.001 denote significant differences compared with (a) adhesion to resting NHBEC at the corresponding time or (b or c) adhesion in the presence of MOPC21.

Stimulation of Eosinophils with C5a or PMA Increases Their Adhesion to NHBEC

To investigate whether inflammatory mediators known to stimulate eosinophil function could increase eosinophil adhesion to NHBEC, we stimulated eosinophils during the adhesion assay with C5a, PAF, FMLP, or LTB₄. For comparison, we also investigated the effect of the phorbol ester PMA on eosinophil adhesion. Data from one experiment of two similar experiments is shown in Figure 4 and demonstrates that adhesion, measured at 30 min, to resting or cytokine-activated NHBEC was increased in the presence of PMA (10⁸ M) or C5a (10⁷ M), but not PAF, FMLP, or LTB₄ (10⁸ or 10⁷ M). The effects of C5a and PMA were therefore studied further. In four experiments, C5a (10⁷ M) significantly (P < 0.05) increased eosinophil adhesion (30 min) to resting NHBEC from 11.4 ± 0.7% to 15.5 ± 0.4% and PMA (10⁸ M) increased adhesion to 20.7 ± 1.7% (P < 0.001). Basal adhesion to activated NHBEC was 11.1 ± 1.3%, and C5a or PMA significantly (P < 0.01) increased this to 21.9 ± 1.0% or 27.6 ± 1.9%, respectively. Eosinophil adhesion to activated NHBEC was also measured at 15, 60, or 120 min; in addition to the lack of effect seen at 30 min (Figure 4), no increase in adhesion could be detected with PAF, LTB₄, or FMLP at these times (Figure 5). In contrast, C5a significantly increased eosinophil adhesion compared with the corresponding basal adhesion at 15, 30, and 60 but not 120 min (Figure 5). PMA also significantly increased eosinophil adhesion to activated NHBEC at 15, 30, 60, or 120 min (data not shown).

View larger version (24K): [in this window] [in a new window]   Figure 4.   Effects of inflammatory mediators or PMA on eosinophil adhesion to resting or activated NHBEC, measured at 30 min. NHBEC were pretreated with culture medium (open bar) or TNF- (1 ng/ml) + IFN- (10 ng/ml; filled bar) for 24 h. Eosinophils were stimulated during the adhesion assay (30 min) with C5a, PAF, FMLP, LTB4, and the phorbol ester PMA at the molar concentrations indicated on the figure, or incubated in the presence of KRPD buffer alone. Results are expressed as percent adhesion, and data are means ± SD of three replicate measurements shown as a representative of two similar experiments.

View larger version (23K): [in this window] [in a new window]   Figure 5.   Effects of inflammatory mediators on eosinophil adhesion to activated NHBEC, measured at 15, 30, 60, or 120 min. NHBEC were pretreated with TNF- (1 ng/ml) + IFN- (10 ng/ ml) for 24 h. Eosinophils were stimulated during the adhesion assay with C5a (107 M), PAF (107 M), FMLP (107 M), or LTB4 (107 M), or incubated in the presence of KRPD buffer alone. Results are expressed as percent adhesion and data are means ± SD of three replicates measurements shown as a representative of two similar experiments. All standard errors are less than 10% but, to improve clarity, only error bars for KRPD buffer or C5a have been included on the figure. *P < 0.01 or **P < 0.001 denote significant differences compared with adhesion to resting NHBEC at the corresponding time.

C5a or PMA Trigger a CD18/ICAM-1-dependent Increase in Eosinophil Adhesion to Cytokine-activated NHBEC

Monoclonal antibodies against CD18 (6.5E; 20 µg/ml), VLA-4 (2B4; 20 µg/ml), or ICAM-1 (BBIG-I1; 20 µg/ml) were used to investigate the role of these CAM in mediating C5a- or PMA-stimulated eosinophil adhesion to NHBEC, measured at 30 min. Although coincubation with 6.5E significantly (P < 0.001) reduced adhesion of PMA-stimulated eosinophils to resting NHBEC to levels detected with unstimulated eosinophils, the response to C5a was not affected. In contrast, 6.5E reduced C5a- and PMA-stimulated eosinophil adhesion to TNF-/IFN--activated NHBEC (Figure 6b). Coincubation with 2B4 had no effect on C5a-stimulated eosinophil adhesion to resting or activated NHBEC (Figure 6), although we have previously shown that this mAb decreases eosinophil adhesion to HLMVEC (48, 49).

View larger version (26K): [in this window] [in a new window]   Figure 6.   Effects of mAb against CD18 (6.5E) or VLA-4 (2B4) on C5a- or PMA-stimulated eosinophil adhesion to (a) resting NHBEC or (b) NHBEC pretreated for 24 h with TNF- (1 ng/ml) + IFN- (10 ng/ml). Eosinophils were incubated (30 min) with NHBEC in the presence of (1) KRPD buffer; (2) C5a (107 M) with 20 µg/ml MOPC21, 6.5E, or 2B4; or (3) PMA (108 M) with MOPC21 or 6.5E. Results are expressed as percent adhesion and are means ± SEM from 5 to 13 experiments. *P < 0.05, **P < 0.01 or ***P < 0.001 denote significant differences compared with KRPD buffer; #denotes a significant difference (P < 0.001), and ns denotes no significant difference, compared with adhesion in the presence of MOPC21.

To determine whether CD18-dependent adhesion was also dependent on ICAM-1, we investigated the effect of an anti-ICAM-1 mAb (BBIG-I1) on C5a- or PMA-stimulated eosinophil adhesion. BBIG-I1 reduced, in part, PMA-stimulated eosinophil adhesion to resting NHBEC, but did not alter C5a-stimulated adhesion (Figure 7a). In contrast, BBIG-I1 inhibited adhesion of PMA- or C5a-stimulated eosinophils to activated NHBEC (Figure 7b) to levels measured on resting NHBEC in the presence of BBIG-I1 (Figure 7a). For example, adhesion of PMA-stimulated eosinophils to resting and activated NHBEC in the presence of BBIG-I1 was 22.3 ± 1.4% and 22.9 ± 1.0%, respectively. These data suggest that CD18/ ICAM-1 is predominantly a pathway mediating eosinophil adhesion to activated, rather than resting, NHBEC.

View larger version (21K): [in this window] [in a new window]   Figure 7.   Effects of mAb against ICAM-1 (BBIG-I1) on C5a- or PMA-stimulated eosinophil adhesion to (a) resting NHBEC or (b) NHBEC pretreated for 24 h with TNF- (1 ng/ml) + IFN- (10 ng/ml). Eosinophils were incubated (30 min) with NHBEC in the presence of (1) KRPD buffer; (2) C5a (107 M) with F(ab')2 fragments of MOPC21 or BBIG-I1 (10 µg/ml); or (3) PMA (108 M) with MOPC21 or BBIG-I1. Results are expressed as percent adhesion and are means ± SEM from four to five experiments. *P < 0.05, **P < 0.01, or ***P < 0.001 denote significant differences compared with KRPD buffer; #P < 0.05 and #P < 0.01 denote a significant difference compared with adhesion in the presence of the respective MOPC21 control.

Eotaxin, But Not RANTES or MIP-1, Increases Eosinophil Adhesion to Cytokine-activated NHBEC in a CD18/ICAM-1-dependent Manner

The effects on eosinophil adhesion of the C-C chemokines eotaxin, RANTES, and MIP-1, which are known to activate eosinophil function, were also investigated. Eotaxin at 30 or 100 but not 10 ng/ml significantly increased eosinophil adhesion to TNF-/IFN--activated NHBEC (Figure 8a). In contrast, eotaxin had no effect on eosinophil adhesion to resting NHBEC (Figure 8a). Neither RANTES nor MIP-1 (30, 100 ng/ml) increased eosinophil adhesion to resting or cytokine-activated NHBEC (Table 2). Finally, 6.5E or BBIG-I1 abolished the eotaxin (100 ng/ml)-induced increase in eosinophil adhesion to cytokine-activated NHBEC (Figures 8b and 8c).

View larger version (16K): [in this window] [in a new window]   Figure 8.   Adhesion of eotaxin-stimulated eosinophils to NHBEC and effects of anti-CD18 (6.5E) or anti-ICAM-1 (BBIG-I1) mAb. NHBEC were pretreated with culture medium (open circle) or with TNF- (1 ng/ml) + IFN- (10 ng/ml) for 24 h (a, filled circle; b and c). Eosinophils were stimulated during the adhesion assay (30 min) with (a) eotaxin (10, 30, and 100 ng/ml), (b) eotaxin (30 ng/ml) in the presence of MOPC21 (20 µg/ml) or 6.5E (20 µg/ml), and (c) eotaxin (30 ng/ml) with F(ab')2 fragments of MOPC21 or BBIG-I1 (10 µg/ ml). To assess basal adhesion, eosinophils were incubated with resting or activated NHBEC in the presence of KRPD buffer. Results are expressed as percent adhesion and data are means ± SEM from four (a and b) or three (c) experiments. *P < 0.01 or **P < 0.001 denote significant differences compared with basal adhesion and #P < 0.01 or ##P < 0.001 compared with the MOPC21 control antibody.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study shows that adhesion pathways dependent on CD18 and ICAM-1 mediate C5a-, eotaxin-, and PMA-stimulated eosinophil adhesion to cytokine-activated NHBEC. To our knowledge, this is the first report to show that C5a or eotaxin increases human eosinophil adhesion to NHBEC and that a CD18/ICAM-1-dependent pathway plays a role in eosinophil adhesion to airway epithelial cells under inflammatory conditions. In the first part of the study, we determined ICAM-1 expression on resting and cytokine-activated NHBEC in our culture system. TNF- and IFN- were chosen because increased levels of these cytokines have been detected in asthmatic BAL fluid and/ or cultured BAL leukocyte supernatant (18, 19). Incubation with IFN- increased ICAM-1 from a low constitutive level of expression and TNF- synergistically enhanced IFN--induced expression, although alone it had no effect. IFN- has previously been shown to induce ICAM-1 expression selectively on human tracheal epithelial cells (16), and TNF- has been shown not only to increase IFN--induced expression on a bronchial epithelial cell line (BEAS-2B) but also to be effective alone (11). Therefore, in the present study we stimulated NHBEC monolayers (24 h) with a combination of IFN- and TNF- to investigate eosinophil-epithelial adhesion under conditions that may resemble those in asthmatic airways.

Basal adhesion of unstimulated eosinophils to resting NHBEC was independent of CD18, ICAM-1, and VLA-4. We were also unable to inhibit basal adhesion with maltose-1-phosphate (Burke-Gaffney and Hellewell; unpublished observation), a phosphorylated disaccharide that inhibited carbohydrate-mediated neutrophil adhesion to A549 cells (50). These results suggest interactions between ligands known to date to be expressed on eosinophils and airway epithelium are unlikely to mediate basal eosinophil adhesion to resting NHBEC. Adhesion of unstimulated eosinophils either to resting or activated NHBEC increased with time; however, such an increase was not specific for eosinophils since neutrophil adhesion also increased. A time-dependent increase in adhesion may be characteristic of epithelial but not endothelial cells because adhesion to A549 epithelial cells, but not HLMVEC, also increased with time. This may provide evidence that mechanism(s) exist to facilitate eosinophil retention in respiratory airways but not blood vessels, where such mechanisms would be inappropriate. Adhesion of C5a-stimulated eosinophils to NHBEC also increased with time and increasing basal adhesion may serve to amplify the effect seen with C5a. Alternatively, C5a may provide a rapid adhesive response that NHBEC may sustain without further eosinophil activation as adhesion at 120 min was similar for unstimulated and C5a-stimulated eosinophils.

We also showed that adhesion of unstimulated eosinophils, measured at 90 or 120 min, was greater to activated than resting NHBEC. Migration of eosinophils across the monolayer is unlikely to account for this difference as fluorescence associated with resting or activated NHBEC, after washing to remove adherent eosinophils, was similar (less than 5% of total added). mAb against CD18 and ICAM-1 abolished the increased adhesion to activated NHBEC, which provides evidence for a role of a CD18/ ICAM-1-dependent pathway in eosinophil adhesion to cytokine-activated NHBEC. These results suggests that, in the absence of an added stimulus, upregulation/activation of eosinophil CAM may occur. Soluble mediators (e.g., eotaxin and granulocyte macrophage colony-stimulating factor) that may be produced in eosinophil/NHBEC cocultures could contribute to eosinophil activation. In support of this, eosinophil cationic protein is released in cocultures of resting cells as early as 2 h (39). Major basic protein, another eosinophil granule protein, but not ECP, has been shown to enhance expression of neutrophil CD11b/CD18 (51), but whether either of these alter eosinophil CD18 integrin expression is not known. Alternatively, the eosinophil ligands that mediate basal adhesion may activate CD18 integrins in a manner similar to that described for L-selectin or CD43 on neutrophils or T cells, respectively (52, 53).

Our results with C5a-, eotaxin-, and PMA-stimulated eosinophil adhesion to cytokine-activated NHBEC provide further evidence for an involvement of a CD18/ ICAM-1-dependent pathway in eosinophil adhesion. Effects of anti-CAM antibodies on C5a- or eotaxin-induced eosinophil adhesion to NHBEC have not previously been investigated, although a recent study showed that mAb against CD18, but not ICAM-1, blocked PMA-stimulated eosinophil adhesion to A549 treated with TNF- to express ICAM-1 (13). It is not clear why our results differ from those of Godding and colleagues (13) unless they reflect differences in the epithelial cells or anti-ICAM-1 mAb used. In our study we used F(ab')₂ fragments of BBIG-I1, whereas Godding and colleagues used F(ab')₂ fragments of R6.5 (13). However, because the latter has been shown to partly inhibit neutrophil adhesion to bronchial epithelial cells (38), the difference in our data compared with that of Godding and colleagues may be due to the epithelial cells used. To our knowledge, a CD18/ ICAM-1-dependent pathway for eosinophil-epithelial adhesion has only been identified to date for PMA-stimulated eosinophil adhesion to RSV-infected A549. Although there is evidence from neutrophil studies that airway epithelial cells may express a non-ICAM-1 ligand for CD18 (10, 36), and IL-5 has been shown to cause a CD18-dependent/ICAM-1-independent increase in eosinophil adhesion to HBEC (39), our results suggest that a CD18/ ICAM-1-dependent pathway plays an important role in chemoattractant-stimulated eosinophil adhesion to activated NHBEC.

In contrast to our results with activated NHBEC, eosinophil adhesion to resting NHBEC was largely independent of a CD18/ICAM-1-dependent pathway. PMA-induced adhesion was dependent on CD18 but only partially dependent on ICAM-1. A study by Godding and colleagues with A549 showed that an anti-CD18, but not an anti-ICAM-1, mAb inhibited PMA-induced adhesion (13). A lack of effect or partial effect of anti-ICAM-1 may suggest a non-ICAM-1 ligand for CD18 on epithelial cells. Alternatively, the inhibitory effects of anti-ICAM-1 may be masked/ reduced when eosinophils are aggregated and PMA is known to be a potent activator of eosinophil aggregation (54).

That C5a-induced eosinophil adhesion to resting NHBEC was not blocked with any of the mAb used in this study is perhaps more intriguing. C5a was the only eosinophil active mediator, other than eotaxin, that increased eosinophil adhesion in our study. The mechanisms responsible for C5a-mediated adhesion to resting NHBEC are the subject of further research as this may have implications for the pathogenesis of asthma. Indeed, complement is thought to play an important role in virus-induced asthma (29). Binding of complement components to epithelial cells has been described in vitro and in vivo during infection with RSV and in this way may activate eosinophils and increase adhesion (29). That eotaxin does not induce adhesion to unactivated NHBEC may suggest a difference between the capacity of these chemoattractants to activate eosinophils.

It is not clear why other eosinophil active mediators that we have previously shown to be active in chemotaxis assays (55) did not increase eosinophil adhesion to NHBEC in this study. PAF has previously been shown to increase (2.5-fold) adhesion to BEAS-2B when eosinophils were preincubated with PAF (30 min) before the adhesion assay (56), so it is possible that this preincubation may enhance the adhesive effect. Responses to FMLP and PAF may also require eosinophils to be primed, as a 30-min exposure to granulocyte macrophage colony-stimulating factor caused a potentiation of PAF- and FMLP-induced adherence to gelatin-coated plastic (25). It is unlikely, however, that the lack of response to FMLP, PAF, and LTB₄ in this study was time dependent, as we were unable to measure an increase in adhesion at 7.5, 15, 30, 60, or 120 min. RANTES has been shown to increase eosinophil adhesion to culture plates coated with soluble ICAM-1 (57), although we were previously unable to demonstrate an increase in adhesion of RANTES- or MIP-1-stimulated eosinophils to HLMVEC activated with TNF- to express ICAM-1 (48). This may suggest that either (1) adhesion to coated plates may be facilitated by a higher concentration of ligand than that expressed on cell monolayers or (2) inhibitory factors associated with cell monolayers, but not artificial surfaces, may act to limit or prevent adhesion stimulated by less potent mediators. The differences we have highlighted between the effects of eotaxin and RANTES on eosinophil adhesion in vitro, may partly explain our previous finding that eotaxin, but not RANTES, induced significant recruitment of ¹¹¹In-eosinophils in vivo in mouse skin (58).

In conclusion, we have shown that C5a, eotaxin, and PMA increase eosinophil adhesion to NHBEC and that adhesion to cytokine-activated NHBEC was dependent on CD18 and ICAM-1. Our results may suggest a role for C5a in asthma that has not previously been reported and strengthen the evidence for a unique involvement of eotaxin amongst the C-C chemokines. Our results also highlight an involvement of a CD18/ICAM-1-dependent pathway for eosinophil adhesion to NHBEC. Thus, these results contribute to our understanding of the factors responsible for the eosinophil-epithelial interactions that facilitate eosinophil retention in the airways of asthmatics and may have important implications for the treatment of asthma.