Asthma is characterized by an airway inflammatory infiltrate that is rich in eosinophilic leukocytes. Cellular fibronectin and VCAM-1, ligands for α4 integrins, are enriched in the fluid of airways of allergic patients subjected to Ag challenge. We therefore hypothesized that ligands of α4 integrins can promote eosinophil survival independent of cell adhesion. Cellular fibronectin and VCAM-1 increased viability of human peripheral blood eosinophil in a dose- and time-dependant manner whether the ligand was coated on the culture well or added to the medium at the beginning of the assay. Eosinophils cultured with cellular fibronectin were not adherent to the bottom of culture wells after 3 days. Treatment with mAb Fib 30 to β7, but not mAb P4C10 or TS2/16 to β1, increased eosinophil survival. The increased survival of eosinophils incubated with Fib 30 was blocked by Fab fragments of another anti-β7 mAb, Fib 504. Eosinophils incubated with soluble cellular fibronectin or mAb Fib 30 for 6 h demonstrated a higher level of GM-CSF mRNA than eosinophils incubated with medium alone. Addition of neutralizing mAb to GM-CSF during incubation, but not mAbs to IL-3 or IL-5, reduced the enhancement of eosinophil survival by soluble cellular fibronectin or mAb Fib 30 to control levels. Thus, viability of eosinophils incubated with cellular fibronectin or VCAM-1 is due to engagement, probably followed by cross-linking, of α4β7 by soluble ligand (or mAb) that stimulates autocrine production of GM-CSF and promotes eosinophil survival.

Asthma and several allergic diseases are characterized by increased numbers of eosinophils and inflammatory mediators (1). Eosinophils are found in increased numbers in the bronchoalveolar lavage (BAL)4 fluid of patients with asthma (1) and are recruited to the airway following challenge with allergenic Ags (2). VCAM-1 (CD106) (3) is increased in the vasculature of trachea in mice after allergen challenge (4). The soluble form of VCAM-1 is increased in the airway of human subjects after allergen challenge, and this increase correlates with increased numbers of eosinophils in the airway (5). Fibronectin levels in BAL fluid are also significantly increased 48 h following Ag challenge, and fibronectin concentrations in BAL fluid strongly correlate with numbers of eosinophils (2).

Eosinophils undergo diapedesis and interact with their environment via cell surface integrins (4). Integrins are heterodimers of α and β subunits that noncovalently associate to form receptors for extracellular matrix proteins and certain cell surface ligands (3). Eosinophils express the integrin α4β1 (very late Ag-4; CD49d/CD29), which binds VCAM-1 and fibronectin (3). The α4 integrin subunit also associates with the β7 subunit on eosinophils, that also binds VCAM-1 and fibronectin (6) and an additional ligand, mucosal addressin cell adhesion molecule-1 (3). The high affinity binding site for α4 integrins on fibronectin is located within the variably spliced region (3). Cells within tissues produce a distinct splice variant of fibronectin that contains the variable region in both subunits, resulting in an additional binding site for α4 integrins and two extra domains, A and B (7). The increase in soluble fibronectin within the airway of challenged subjects contains the extra domain (ED)-A region and, thus, is at least in part the cellular form of fibronectin (2). Several recent studies have demonstrated the importance of α4β1 in several inflammatory diseases, including contact hypersensitivity and responses to lung Ag challenge in animal models, using function-blocking Abs against α4β1 (8).

Previous studies of eosinophil viability have tested fibronectin adsorbed to surfaces, thus mimicking the state of proteins after deposition in extracellular matrix (9, 10, 11). To determine the activities of VCAM-1 and cellular fibronectin in the airway, we tested the ability of these soluble ligands for α4 integrins to affect eosinophil viability in vitro. Both cellular fibronectin and recombinant soluble VCAM-1 (rsVCAM-1) increased viability of eosinophils when incubated in solution during the assay, whereas plasma fibronectin did not. Engagement of α4β7 with mAb Fib 30 increased eosinophil viability in the absence of additional ligands. Levels of message for GM-CSF, a cytokine known to enhance eosinophil viability (12), was increased in eosinophils incubated with cellular fibronectin or Fib 30. In addition, Abs blocking GM-CSF, but not IL-3 or IL-5, inhibited the survival of eosinophils incubated with cellular fibronectin or Fib 30, indicating that GM-CSF generation was responsible for this effect. Thus, eosinophil survival is enhanced by soluble ligands for α4β7 via autocrine production of GM-CSF.

Low endotoxin sterile water was purchased from Baxter Healthcare (Deerfield, IL), and care was taken not to introduce endotoxin contamination into any reagents. HBSS containing calcium and magnesium, RPMI 1640 medium, l-glutamine, penicillin, streptomycin, and low endotoxin heat-inactivated FCS were purchased from Life Technologies (Grand Island, NY). Anti-CD16-conjugated magnetic beads were obtained from Miltenyi Biotech Inc. (Sunnyvale, CA). Percoll, fluorescein diacetate, propidium iodide, ionomycin, and polymyxin B were purchased from Sigma (St. Louis, MO). Hoechst 33342 apoptotic stain was bought from Molecular Probes (Eugene, OR). Recombinant human IL-5 and GM-CSF were purchased from R&D Systems (Minneapolis, MN).

Hybridoma cell lines producing mAbs to β1 (TS2/16, Ref. 13), to β7 (Fib 504 and Fib 21, Ref. 14), and to β2 (TS1/18, Ref. 13) were purchased from American Type Culture Collection (ATCC, Manassas, VA). mAbs were purified from serum-free (HyQ CCM1, HyClone, Logan, UT) tissue culture supernatant by passage over a column of protein G complexed to agarose (Life Technologies), eluted with 50 mM glycine-HCl, pH 2.5, and immediately buffer exchanged to 20 mM phosphate, 10 mM EDTA, pH 7.0. Fab fragments were prepared using the Fab preparation kit from Pierce (Rockford, IL) according to the manufacturer’s instructions. Purified intact and Fab fragments were concentrated using Centriplus spin concentration filters of 100 kDa or 10 kDa m.w. cut off, respectively (Amicon, Beverely, MA). Additional mAbs to β7 (Fib 21, Fib 22, Fib 27, and Fib 30) were a generous gift from David P. Andrew (Amgen, Boulder, CO). mAbs P4C10 to β1 and P4C2 to α4, and mAbs to the cytokines IL-3, IL-5, and GM-CSF were purchased from R&D Systems. mAb HP2/4 was a gift from Dr. Francisco Sánchez-Madrid, Universidad Autónoma de Madrid, Madrid, Spain (15). Control, nonspecific mouse and rat mAbs were purchased from Sigma and Life Technologies.

The seven-domain form of VCAM-1, lacking the intracellular region (rsVCAM-1) and purified by immunoaffinity chromatography from the conditioned medium of transfected COS cells, was a generous gift from Dr. Roy R. Lobb (Biogen Research Corporation, Cambridge MA) (16). Additional supplies were purchased from R&D Systems. Recombinant soluble ICAM-1 was a gift from Dr. Robert Rothlein (Boehringer Ingelheim, Ridgefield, CT). Type 1 laminin, from the Engelbreth-Holm-Swarm mouse tumor, was a gift from Dr. Hynda Kleinman (National Institutes of Health, Bethesda, MD).

Human plasma fibronectin was a gift from Armour Pharmaceutical (Tuckahoe, NY). The peptide EILDVPST, containing the high affinity binding site in fibronectin for α4 integrins, and a nonbinding control peptide (EILEVPST) were obtained from Peninsula Laboratories (Belmont, CA). Cellular fibronectin was purchased from Sigma or purified by gelatin affinity chromatography (Sigma) from conditioned media of AH-1F human foreskin fibroblasts using conditions to minimize exposure to endotoxin (17). Purified protein migrated as bands of 240 kDa on reducing SDS-PAGE and about 400 kDa on nonreducing SDS-PAGE and reacted by immunoblotting with the IST-9 mAb (18) against the ED-A module of cellular fibronectin.

Fibronectin preparations were examined for potentially confounding contaminants using the Limulus amebocyte lysate assay for endotoxin (E-Toxate, Sigma) or by direct ELISA for cytokines. To estimate the concentration of endotoxin contamination in protein solutions, several dilutions of the stock protein solutions were tested according to the manufacturer’s instructions and compared with a standard endotoxin solution. Protein solutions were boiled for 2 min before the assay to eliminate false positive results that may result from the presence of trace contaminants of serine proteases. In these assays, 1 endotoxin unit (EU) represented about 2 ng of purified LPS. The smallest concentration of LPS standard detectable in the assay was between 0.03 and 0.1 EU/ml. At the highest protein concentrations used in the survival assays, plasma fibronectin contained fewer than 0.03 EU/ml, cellular fibronectin preparations contained fewer than 0.03 EU/ml to 1 EU/ml, and rsVCAM-1 contained 0.5 EU/ml. Polymyxin B was present in all assays to further minimize the effect of endotoxin. A sensitive two step sandwich ELISA was used to detect cytokines contamination in the cellular fibronectin preparation (19). Abs against IL-5, GM-CSF, IFN-γ, and RANTES were purchased from PharMingen (San Diego, CA). The signal from samples containing cellular fibronectin at the highest concentration used in the survival assays was below the limit of detection (3 pg/ml) for the four cytokines tested.

Eosinophils were isolated from the peripheral blood of normal donors and of patients with allergic rhinitis or mild bronchial asthma by an anti-CD16 magnetic bead cell separation system (MACS; Miltenyi Biotech), as described by Hansel et al. (20). Heparinized venous blood was centrifuged over a Percoll gradient (1.090 g/ml), and RBC were removed by hypotonic lysis of the resulting granulocyte pellet. The cells were then washed twice with HBSS without Ca2+ supplemented with 2% FCS and incubated 40 min at 40°C with 100 μl anti-CD16 mAb-conjugated magnetic beads. Eosinophils were negatively selected by passing the CD16-labeled granulocytes over a MACS column in a magnetic field. The purity of eosinophils (Diff-Quick stains, Baxter Healthcare Corporation, McGraw Park, IL) was at least 95% in all experiments and was greater than 99% in most instances. Contaminating cells were predominantly neutrophils. Viability was greater than 99% as determined by trypan blue dye exclusion.

Ninety six-well flat-bottom tissue culture-treated plates (Corning, Corning, NY) were coated with 100 μl protein solutions overnight at 40°C. Residual protein was decanted from wells, and nonspecific protein-binding sites in the wells were blocked with 100 μl neat FCS for 2 h at 37°C. Alternatively, proteins were added concurrently with cells to wells that had been blocked with neat FCS. The wells were washed 3× with HBSS, and purified eosinophils (1 to 2 × 105/0.1 ml) in RPMI 1640 containing 1% FCS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, and 50 U/ml polymyxin B were added to each well. In some experiments, eosinophils were incubated for 30 min at 4°C with mAbs to integrins or cytokines as noted. mAbs were not removed during the assay. Eosinophils were warmed briefly to 37°C before addition of proteins used to promote survival, as noted. Eosinophils were incubated at 37°C in a humidified 5% CO2 incubator for up to 3 days, when viability was determined. In some experiments, spent medium containing anti-integrin mAbs was harvested, centrifuged to remove any remaining cells, and stored at −70°C for further analysis. Sufficient functional mAb remained at the end of the assay to maximally stain fresh eosinophils by flow cytometry and to inhibit eosinophil adhesion to rsVCAM-1 (not shown). At the end of the 3-day incubation in some experiments, nonadherent eosinophils were transferred to other wells on the plate, and the original wells were rinsed twice with 50 μl HBSS to remove any remaining nonadherent cells. The rinses were added to the wells containing nonadherent cells. HBSS (100 μl) was added to wells containing the remaining adherent cells. Duplicate, unwashed wells were examined to compare the percentage viability in the whole cell population.

Viability of eosinophils cultured with matrix proteins has been measured in medium containing 2% or no serum for 3 days (9, 10) or medium containing 10% serum for 5 days (21). We found that eosinophils incubated in RPMI 1640 containing 0, 1, 2, 5, or 10% FCS exhibited similar levels of viability. Eosinophils cultured in medium containing serum were significantly more viable on day 2 than eosinophils incubated without serum, but this difference was not observed on day 3, when viability was less than 10% (data not shown). We chose to culture eosinophils for 3 days in media containing only 1% FCS to reduce effects due to serum. IL-5 (0.1 ng/ml) was added to separate wells as a positive control.

Quantitation of protein coated onto wells after overnight incubation was determined using the BCA protein assay (Pierce). Wells were coated with 20, 10, or 1 μg/0.1 ml of cellular fibronectin, or plasma fibronectin overnight and rinsed three times with HBSS as described above. Amounts of protein remaining in the wells were determined by comparison with a standard curve of BSA.

The amount of cellular fibronectin added in solution that became attached to the well was determined by a direct ELISA. Wells were blocked with FCS as described above, rinsed three times with HBSS and RPMI 1640 containing 1% FCS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, and 50 U/ml polymyxin B with or without plasma or cellular fibronectin at 2.0 μg/0.1 ml was added to each well. After incubation for up to 3 days, medium was removed, and the wells were rinsed twice with TBS containing 0.1% Tween 20. After blocking wells with 1% BSA in TBS, mAb IST-9 (18) (Accurate Chemical, Westbury, NY), against the ED-A domain of cellular fibronectin, was added at 250 ng/ml and incubated for 2 h at room temperature. Wells were rinsed four times with TBS + Tween 20, and secondary alkaline phosphatase-conjugated goat anti-mouse (Boehringer Mannheim, Indianapolis, IN) diluted 1:200 was incubated in each well for 2 h at room temperature. Unbound secondary Ab was removed with four rinses of TBS + Tween 20, and the reaction was developed with alkaline phosphatase substrate 104 (Sigma). Absorbance was measured at 405 nm to calculate the concentration of cellular fibronectin present, as compared with a standard curve of cellular fibronectin coated in TBS + 0.1% BSA, with a lower limit of detection at a coating concentration of 5 ng/ml.

Cell viability was determined after 72 h of incubation (unless otherwise noted) at 37°C in 5% CO2 by staining cells with fluorescein diacetate (5 μg/ml) and propidium iodide (1 μg/ml) (22). Fluorescein diacetate is cleaved by esterases in viable, metabolically active cells to yield fluorescein; thus, viable cells are stained green (23). Propidium iodide is permeable to cells that have compromised membrane integrity; thus, dead cells are stained red. Eosinophils were brought to the bottom of the wells by centrifugation (400 × g for 5 min at 4°C) and were viewed using an inverted microscope with a dual wavelength filter (Diaphot-TMD, Filter:B-2A, Nikon, Japan). At least 100 to 200 cells in three to four wells were counted for each condition tested, and the results were expressed as the mean percentage of viable cells.

To confirm the results obtained by manual counting, eosinophils were analyzed by flow cytometry. After the 3-day incubation (Corning 24-well tissue culture-treated plates), fluorescein diacetate was added as above to stain live eosinophils. In replicate plates, apoptotic cells were stained with the bis-benzimidazole dye for DNA, Hoechst 33342 at 1 μg/ml for 10 min (24). Cells were removed from the wells by pipetting, pelleted by centrifugation, and resuspended in 500 μl PBS + 0.2% BSA and filtered through a 50-μm mesh filter to remove clumps of cells. Propidium iodide, 2.5 μg/ml, was added immediately before analysis. Samples were analyzed on a FACStarPlus (Becton Dickinson) equipped with two lasers: a Coherent argon laser tuned to 488 nm and a Coherent krypton laser set to multiline UV (Becton Dickinson Immunocytometry Systems, San Jose, CA).

Eosinophils (6 × 105) were incubated with control or anti-integrin mAbs for 30 min at 4°C in RPMI 1640 containing 1% FCS, 2 mM l-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 U/ml polymyxin B. In additional experiments, eosinophils were incubated with spent medium from survival assays containing anti-integrin mAbs. Unbound mAb and media were rinsed away with PBS containing 1% BSA and centrifugation. Eosinophils were resuspended in PBS + 1% BSA and incubated with FITC-labeled anti-rat or anti-mouse secondary Abs as appropriate for 40 min on ice in the dark. Cells were rinsed twice, and propidium iodide was added immediately before analysis. Samples were analyzed on a FACStarPlus (Becton Dickinson).

Eosinophils (3 × 106) in 1 ml RPMI 1640 containing 1% FCS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin were either immediately lysed or incubated for 6 h with stimuli before lysis and RNA extraction in Tri reagent (Sigma) according to the manufacturer’s instructions. Quantitative RT-PCR for GM-CSF message was performed as described previously (25). In brief, PCR of the reverse-transcribed message was performed with the following specific primers for GM-CSF: 5′ primer, 5′-ATG TGG CTG CAG AGC CTG CTG C-3′; 3′ primer, 5′-C TGG CTC CCA GCA GTC AAA GGG-3′ (Genset, La Jolla, CA). Southern blotting was performed to verify the identity of PCR products using probes synthesized by PCR of GM-CSF cDNA as described previously (25). The blot was scanned, and band density was determined using Sigmagel blot analysis software (Jandel Scientific, San Raphael, CA). Similarly, quantitation of the housekeeping message, GAPDH, was performed as a control for the reactions. Cytokine message levels were normalized for the amount of GAPDH message detected in each sample by dividing the mRNA units derived from the scanned GM-CSF blot by the units obtained for each sample on the GAPDH blot and are reported as normalized units.

Results are given as mean ± SEM except where otherwise noted. Statistical significance of the differences between various treatments was assessed using Minitab statistical software (State College, PA) one-way ANOVA with Fisher subcommands for multiple comparisons (26). A p value of less than 0.05 was considered significant.

Our initial experiments replicate the finding that eosinophils exhibit extended viability when cultured in containers coated with cellular, but not plasma, fibronectin (9, 10). Cellular fibronectin enhanced eosinophil viability when coated at concentrations above 10.0 μg/0.1 ml whereas similar coatings of plasma fibronectin did not increase eosinophil survival (Fig. 1,A). We then tested rsVCAM-1, like fibronectin a ligand for α4 integrins, and found that wells coated with at least 1.0 μg/0.1 ml rsVCAM-1 increased eosinophil viability (Fig. 1 A).

FIGURE 1.

Effects of integrin ligands on eosinophil survival when coated onto plates or used in solution. Eosinophils were incubated with cellular fibronectin (□), rsVCAM-1 (○), or plasma fibronectin (⋄), either (A) coated onto the wells or (B) added concurrently with the eosinophils in solution at the beginning of the assay at the concentrations indicated. Data are expressed as the mean ± SEM percentage of viability of three experiments. Cellular fibronectin was significantly different (p < 0.05) than no addition (0 μg/0.1 ml) at 10 and 20 μg/0.1 ml in A and 0.1 and 0.2 μg/0.1 ml in B; rsVCAM-1 was significantly different (p < 0.05) than no addition at 10 and 20 μg/0.1 ml in A and 0.1 and 0.2 μg/0.1 ml in B in two of three experiments; and plasma fibronectin was not significantly different from no addition at any concentration tested in A or B.

FIGURE 1.

Effects of integrin ligands on eosinophil survival when coated onto plates or used in solution. Eosinophils were incubated with cellular fibronectin (□), rsVCAM-1 (○), or plasma fibronectin (⋄), either (A) coated onto the wells or (B) added concurrently with the eosinophils in solution at the beginning of the assay at the concentrations indicated. Data are expressed as the mean ± SEM percentage of viability of three experiments. Cellular fibronectin was significantly different (p < 0.05) than no addition (0 μg/0.1 ml) at 10 and 20 μg/0.1 ml in A and 0.1 and 0.2 μg/0.1 ml in B; rsVCAM-1 was significantly different (p < 0.05) than no addition at 10 and 20 μg/0.1 ml in A and 0.1 and 0.2 μg/0.1 ml in B in two of three experiments; and plasma fibronectin was not significantly different from no addition at any concentration tested in A or B.

Close modal

Because lavage fluid from eosinophil-rich allergen-challenged bronchial airway segments contains increased concentrations of sVCAM-1 and cellular (ED-A+) fibronectin (2, 5), we tested whether cellular fibronectin and rsVCAM-1 in solution with the eosinophils at the start of the assay increased eosinophil viability. To compare similar amounts of protein, we determined the amount of protein present on the coated plates using the BCA protein assay (Table I). Approximately 4 μg of protein per well was present after coating with saturating concentrations of protein. Therefore, less was added in solution at the beginning of the assay than used to coat the plates (note scale of x-axis in Fig. 1, A and B). The viability of eosinophils was increased when 1.0 or 2.0 μg/0.1 ml of cellular fibronectin was added in solution at the beginning of the assay (Fig. 1,B). No detectable cellular fibronectin was attached to the bottom of the wells after incubation for up to 3 days as determined by ELISA using the cellular fibronectin-specific mAb IST-9. None of the concentrations of plasma fibronectin used increased viability significantly greater than eosinophils incubated without additional protein (Fig. 1,B), even at concentrations as high as 20 μg/0.1 ml (data not shown). Peptides containing the LDV high affinity binding site for α4 integrins (EILDVPST) did not alter eosinophil viability differently than a nonbinding control peptide (EILEVPST) (18 ± 13% vs 16 ± 6% viability at 0.4 μg/0.1 ml, respectively, n = 3). Similar to soluble cellular fibronectin, concentrations of 1.0 or 2.0 μg/0.1 ml of rsVCAM-1 increased viability of eosinophils (Fig. 1 B), although with less of an effect than when coated onto the bottom of the well. Type 1 laminin, a ligand for α6β1, did not increase eosinophil viability when coated (20 ± 5% vs 17 ± 1% viability in uncoated wells, n = 3) or when added in solution (16 ± 5% vs 10 ± 3% viability with no addition, n = 3). Similarly, soluble ICAM-1, a ligand for β2 integrins, did not alter eosinophil viability when it was added in solution (12% viability vs 10% viability with no addition on average in two experiments).

Table I.

Amount of protein remaining on plates after coatinga

Amount of Protein Coated onto Plate (μg)Amount of Protein Remaining in Wells After Rinsing (μg)
Cellular fibronectinPlasma fibronectin
0.5 0.6 
10 3.3 1.8 
20 4.0 3.7 
Amount of Protein Coated onto Plate (μg)Amount of Protein Remaining in Wells After Rinsing (μg)
Cellular fibronectinPlasma fibronectin
0.5 0.6 
10 3.3 1.8 
20 4.0 3.7 
a

Data are shown as the mean of two wells of one experiment.

Eosinophils were stained with Hoechst 33342, which binds fragmented DNA of apoptotic cells, and counter stained with propidium iodide to stain dead cells, so live cells remain unstained (Fig. 2). Hoechst 33342 staining was compared with staining with fluorescein diacetate (Fig. 2). Percentages of cells permeable to propidium iodide (dead cells) were similar regardless of the other chromophore used. There were not significant differences among percentages of cells stained with Hoechst 33342 but negative for propidium iodide (i.e., apoptotic cells) for any of the conditions tested. Eosinophils incubated for 1 and 2 days also indicated no difference between the amounts of Hoechst 33342+/propidium iodide cells when cultured with medium alone, or medium containing cellular fibronectin, rsVCAM-1, or IL-5 (data not shown).

FIGURE 2.

Viability determinations by staining with fluorescein diacetate or Hoechst 33342. Dot plots of FACS analyzed eosinophils treated with Hoechst 33342 to stain apoptotic cells (Hoechst 33342) or fluorescein diacetate to stain live cells (FDA). Eosinophils were counterstained with propidium iodide (PI) to indicate dead cells. Eosinophils were incubated with media alone (Control), 20 μg/ml cellular fibronectin, 20 μg/ml rsVCAM-1, or 0.1 ng/ml IL-5 for 3 days as described in Materials and Methods. Data shown are eosinophils from replicate wells of each condition stained with either Hoechst 33342 or fluorescein diacetate. The percentage of viability was calculated for the Hoechst 33342 stained cells by addition of the lower left (Hoechst/propidium iodide) and lower right (Hoechst+/propidium iodide) quadrants, and for fluorescein diacetate-stained cells by addition of the lower left (fluorescein diacetate low/propidium iodide) and lower right (fluorescein diacetate+/propidium iodide) quadrants. The percentage of apoptotic cells was determined from the Hoechst+/propidium iodide population. This experiment was repeated with similar results.

FIGURE 2.

Viability determinations by staining with fluorescein diacetate or Hoechst 33342. Dot plots of FACS analyzed eosinophils treated with Hoechst 33342 to stain apoptotic cells (Hoechst 33342) or fluorescein diacetate to stain live cells (FDA). Eosinophils were counterstained with propidium iodide (PI) to indicate dead cells. Eosinophils were incubated with media alone (Control), 20 μg/ml cellular fibronectin, 20 μg/ml rsVCAM-1, or 0.1 ng/ml IL-5 for 3 days as described in Materials and Methods. Data shown are eosinophils from replicate wells of each condition stained with either Hoechst 33342 or fluorescein diacetate. The percentage of viability was calculated for the Hoechst 33342 stained cells by addition of the lower left (Hoechst/propidium iodide) and lower right (Hoechst+/propidium iodide) quadrants, and for fluorescein diacetate-stained cells by addition of the lower left (fluorescein diacetate low/propidium iodide) and lower right (fluorescein diacetate+/propidium iodide) quadrants. The percentage of apoptotic cells was determined from the Hoechst+/propidium iodide population. This experiment was repeated with similar results.

Close modal

Survival of eosinophils incubated with soluble integrin ligands was measured daily for 3 days to quantify the rate of cell death (Fig. 3). After 24 h, nearly all (89 ± 2%) the cells were viable regardless of the matrix protein present. Viability was more variable after 48 or 72 h, when viabilities of cellular fibronectin-, rsVCAM-, and IL-5-treated eosinophil samples were significantly higher than controls (Fig. 3). Viability of eosinophils incubated with plasma fibronectin in solution were not different from controls on any day tested (Fig. 3).

FIGURE 3.

Time course of viability of eosinophils incubated with integrin ligands. Eosinophils were incubated in three replicate wells of each condition in three replicate plates, one plate for counting on each of the days indicated. Eosinophils were counted at the beginning of the assay to determine 100% viability on day 0 as described in Materials and Methods. Eosinophils were incubated with media containing 0.01 ng/0.1 ml IL-5 (▵), 2.0 μg/0.1 ml cellular fibronectin (cFn, □), 2.0 μg/0.1 ml rsVCAM-1 (○), 2.0 μg/0.1 ml plasma fibronectin (pFn, ⋄), or no additions (▿). Data are expressed as the mean ± SEM of three experiments for no addition-, cellular fibronectin-, and IL-5-treated eosinophils; and the average of two experiments of rsVCAM-1-treated eosinophils; and one experiment of plasma fibronectin-treated eosinophils. IL-5-, cellular fibronectin-, and rsVCAM-1-treated eosinophils were significantly different from no addition-treated eosinophils on day 2 and day 3 (p < 0.05).

FIGURE 3.

Time course of viability of eosinophils incubated with integrin ligands. Eosinophils were incubated in three replicate wells of each condition in three replicate plates, one plate for counting on each of the days indicated. Eosinophils were counted at the beginning of the assay to determine 100% viability on day 0 as described in Materials and Methods. Eosinophils were incubated with media containing 0.01 ng/0.1 ml IL-5 (▵), 2.0 μg/0.1 ml cellular fibronectin (cFn, □), 2.0 μg/0.1 ml rsVCAM-1 (○), 2.0 μg/0.1 ml plasma fibronectin (pFn, ⋄), or no additions (▿). Data are expressed as the mean ± SEM of three experiments for no addition-, cellular fibronectin-, and IL-5-treated eosinophils; and the average of two experiments of rsVCAM-1-treated eosinophils; and one experiment of plasma fibronectin-treated eosinophils. IL-5-, cellular fibronectin-, and rsVCAM-1-treated eosinophils were significantly different from no addition-treated eosinophils on day 2 and day 3 (p < 0.05).

Close modal

We examined whether the viable eosinophils were those that became adherent during assay. When viewed daily, only a minority of eosinophils were adherent when plates were examined by phase microscopy after gentle swirling. Representative photographs of wells containing adherent, nonadherent, or unwashed (total) eosinophils that had been incubated with medium with no additions, cellular fibronectin, or IL-5 are shown in Fig. 4,A. Nearly all of the eosinophils were nonadherent at the end of the assay, as noted by the decreased numbers of cells in the washed wells (Fig. 4,A, Adherent). The majority of the cells metabolically able to cleave fluorescein diacetate were found in the nonadherent population, as shown by the number of green live cells compared with the red dead cells (Fig. 4,A, Nonadherent). A higher proportion of the nonadherent cells was viable than the adherent cells (Fig. 4 B), indicating that prolonged adhesion of the eosinophils to the bottom or sides of the wells was not necessary.

FIGURE 4.

Effect of adherence to the bottom of the well in the viability assay. Eosinophils incubated in the presence of medium containing no addition (Control), 2.0 μg/0.1 ml cellular fibronectin (cFn), or 0.01 ng/0.1 ml IL-5 (IL-5) were examined after 3 days of incubation for viability of adherent and suspended cells. Wells were rinsed to remove nonadherent cells as described in Materials and Methods. A, Photographs of representative fields of each condition from one experiment. Live cells are stained green with fluorescein diacetate, and dead cells are stained red with propidium iodide. B, Graphical representation of the data from four independent experiments. Eosinophils were incubated with media containing no addition (Control), 2.0 μg/0.1 ml cellular fibronectin (cFn), or 0.01 ng/0.1 ml IL-5 (IL-5) for 3 days as described in Materials and Methods. Eosinophils remaining within the culture well after the washes (Adherent, shaded hatched bars), eosinophils collected from rinsing the wells (Nonadherent, filled bars), and wells that were not rinsed (Total, hatched bars) were counted for the percentage of the total number of cells counted in each well that were viable on day 3. Data are expressed as the mean ± SEM of four experiments of viability counts of adherent, nonadherent, and total cells.

FIGURE 4.

Effect of adherence to the bottom of the well in the viability assay. Eosinophils incubated in the presence of medium containing no addition (Control), 2.0 μg/0.1 ml cellular fibronectin (cFn), or 0.01 ng/0.1 ml IL-5 (IL-5) were examined after 3 days of incubation for viability of adherent and suspended cells. Wells were rinsed to remove nonadherent cells as described in Materials and Methods. A, Photographs of representative fields of each condition from one experiment. Live cells are stained green with fluorescein diacetate, and dead cells are stained red with propidium iodide. B, Graphical representation of the data from four independent experiments. Eosinophils were incubated with media containing no addition (Control), 2.0 μg/0.1 ml cellular fibronectin (cFn), or 0.01 ng/0.1 ml IL-5 (IL-5) for 3 days as described in Materials and Methods. Eosinophils remaining within the culture well after the washes (Adherent, shaded hatched bars), eosinophils collected from rinsing the wells (Nonadherent, filled bars), and wells that were not rinsed (Total, hatched bars) were counted for the percentage of the total number of cells counted in each well that were viable on day 3. Data are expressed as the mean ± SEM of four experiments of viability counts of adherent, nonadherent, and total cells.

Close modal

Flow cytometry was used to assess expression levels of integrins on eosinophils and also to compare the relative affinities of the rat anti-mouse β7 mAbs (Table II). Eosinophils stained strongly with anti-β2 mAb TS1/18 (Table II). The level of β1 staining with P4C10 was slightly lower than staining with anti-α4 mAb HP2/4. Eosinophils also stain positively with the β1-activating mAb TS2/16 (Table II). The anti-β7 mAbs ranked in order of strongest to weakest staining as Fib 30 > Fib 27 > Fib 21 > Fib 22 (Table II). Eosinophils stained stronger with anti-β7 mAb Fib 504 than Fib 21 (Table II). Fib 22 staining was not different from control rat mAb and has previously been shown not to cross-react with human β7 integrins (14), and so was used as a control in survival experiments.

Table II.

Flow cytometry of anti-integrin mAbs on eosinophils

mAbGeometric Mean Fluorescent Intensity
Expt. 1Expt. 2Expt. 3
Mouse IgG 2.8 3.3 33.7 
Rat IgG 4.1 2.6 32.3 
HP2/4 (mouse anti-α4ND 17.5 ND 
P4C10 (mouse anti-β115.8 16.0 ND 
TS2/16 (mouse anti-β1ND ND 94.8 
Fib 21 (rat anti-β75.9 6.7 32.3 
Fib 22 (rat anti-β73.6 3.2 ND 
Fib 27 (rat anti-β78.6 8.9 ND 
Fib 30 (rat anti-β79.3 10.0 ND 
Fib 504 (rat anti-β7ND ND 56.5 
TS1/18 (mouse anti-β2ND 177.2 312.7 
mAbGeometric Mean Fluorescent Intensity
Expt. 1Expt. 2Expt. 3
Mouse IgG 2.8 3.3 33.7 
Rat IgG 4.1 2.6 32.3 
HP2/4 (mouse anti-α4ND 17.5 ND 
P4C10 (mouse anti-β115.8 16.0 ND 
TS2/16 (mouse anti-β1ND ND 94.8 
Fib 21 (rat anti-β75.9 6.7 32.3 
Fib 22 (rat anti-β73.6 3.2 ND 
Fib 27 (rat anti-β78.6 8.9 ND 
Fib 30 (rat anti-β79.3 10.0 ND 
Fib 504 (rat anti-β7ND ND 56.5 
TS1/18 (mouse anti-β2ND 177.2 312.7 

Viability of eosinophils that were pretreated with mAb P4C2 or HP2/4 to α4 or P4C10 to β1 before incubation with cellular fibronectin were not significantly different from untreated eosinophils incubated with cellular fibronectin (Table III). However, a small but significant decrease was observed when the mAbs P4C2 and P4C10 were combined, blocking both α4 and β1 (Table III, 28% average inhibition, n = 2). Because mAbs to α4β1 did not completely block survival of eosinophils incubated with soluble cellular fibronectin, a panel of mAbs to β7 were tested for their ability to decrease eosinophil viability (Fig. 5). Anti-β7 mAbs Fib 21, Fib 22, and Fib 27 did not significantly alter the viability of eosinophils incubated with soluble cellular fibronectin. In contrast, Fib 30 to β7 increased eosinophil survival in the presence of soluble cellular fibronectin (Fig. 5).

Table III.

Effect of anti-integrin mAbs on viability of eosinophils incubated with soluble cellular fibronectina

AdditionsViability of Eosinophils on Day 3 (% of total)
Expt. 1Expt. 2Expt. 3Expt. 4Expt. 5
No addition 13 13 34 
Cellular fibronectin 37 38 34 40 60 
Cellular fibronectin+ anti-α1 mAb P4C10 30 24 39 41 57 
Cellular fibronectin+ anti-α4 mAb P4C2 32 27 34 ND ND 
Cellular fibronectin+ anti-β1 mAb P4C10+ anti-α4 mAb P4C2 ND 24 28 ND ND 
Cellular fibronectin+ anti-α4 mAb HP2/4 ND ND ND 35 69 
AdditionsViability of Eosinophils on Day 3 (% of total)
Expt. 1Expt. 2Expt. 3Expt. 4Expt. 5
No addition 13 13 34 
Cellular fibronectin 37 38 34 40 60 
Cellular fibronectin+ anti-α1 mAb P4C10 30 24 39 41 57 
Cellular fibronectin+ anti-α4 mAb P4C2 32 27 34 ND ND 
Cellular fibronectin+ anti-β1 mAb P4C10+ anti-α4 mAb P4C2 ND 24 28 ND ND 
Cellular fibronectin+ anti-α4 mAb HP2/4 ND ND ND 35 69 
a

Data are shown as the mean of three wells of each experiment.

FIGURE 5.

Effect of mAbs to α4 integrins on survival of eosinophils mediated by cellular fibronectin. Eosinophils were pretreated for 30 min at 40°C with a 1:500 dilution of anti-β1 mAb P4C10 or 0.5 μg/0.1 ml rat anti-mouse β7 mAbs Fib 21, Fib 22, Fib 27, Fib 30, or control mouse IgG before incubation with medium containing no addition (No cFn) or medium containing 2.0 μg/0.1 ml cellular fibronectin. Data are expressed as the mean ± SEM of seven experiments for no addition, cellular fibronectin, Fib 21, Fib 22, Fib 27, and Fib 30; and the mean ± SEM of three experiments for mouse IgG (control mAb) and P4C10. Fib 30 was significantly different (p < 0.05) from no mAb (cellular fibronectin-treated).

FIGURE 5.

Effect of mAbs to α4 integrins on survival of eosinophils mediated by cellular fibronectin. Eosinophils were pretreated for 30 min at 40°C with a 1:500 dilution of anti-β1 mAb P4C10 or 0.5 μg/0.1 ml rat anti-mouse β7 mAbs Fib 21, Fib 22, Fib 27, Fib 30, or control mouse IgG before incubation with medium containing no addition (No cFn) or medium containing 2.0 μg/0.1 ml cellular fibronectin. Data are expressed as the mean ± SEM of seven experiments for no addition, cellular fibronectin, Fib 21, Fib 22, Fib 27, and Fib 30; and the mean ± SEM of three experiments for mouse IgG (control mAb) and P4C10. Fib 30 was significantly different (p < 0.05) from no mAb (cellular fibronectin-treated).

Close modal

We tested dose curves of several mAbs to integrins, to mimic divalent ligands, for their ability to promote eosinophil survival in the absence of cellular fibronectin or other survival stimuli. The β7 mAb Fib 30 significantly increased survival of eosinophils without additional ligands (Fig. 6). Fib 30 also enhanced viability when the mAb was coated onto the bottom of culture wells at 0.5 μg/0.1 ml (data not shown). Other anti-β7 mAbs increased survival, although at much lower levels. Fib 21 and Fib 22 increased viability to 26 ± 3% (n = 4) and 23 ± 3% (n = 3), respectively. The maximum viability of eosinophils incubated with Fib 27 (27 ± 4%, n = 6) was not statistically significantly greater than companion samples incubated without added mAb. The mAb TS2/16, which changes the β1 integrin into an active structural conformation (27), also did not increase eosinophil viability (Fig. 6). In one experiment, the anti-β2 integrin TS1/18 tested in a similar dose curve did not increase eosinophil survival (maximum of 12% viability). Similarly, mAbs HP2/4 and P4C10 to the α4 integrin subunit did not increase eosinophil viability (maximum viability 6% and 5%, respectively). Cleavage of the Fib 30 mAb into monovalent Fab fragments eliminated the survival-enhancing effect of this mAb (24 ± 9% maximum viability vs 12 ± 3% viability in controls, n = 4). Comparatively, maximum survival of eosinophils in the presence of Fab fragments of Fib 21 or TS2/16 were equally low (Fib 21 Fab, an average of 24%, n = 2; and TS2/16 Fab, 16 ± 9%, n = 3).

FIGURE 6.

Effect of engagement of α4β1 or α4β7 integrins by mAbs on the survival of eosinophils. Eosinophils were incubated for 3 days with the indicated concentrations of the mAbs Fib 30, TS2/16, or Fab fragments of mAb Fib 30. Data are expressed as the mean ± SEM of four separate experiments for mAb Fib 30 and Fab fragments of Fib 30, and three experiments for TS2/16. Fib 30 was significantly different (p < 0.05) from no mAb addition (0 μg/0.1 ml) at concentrations greater than 0.1 μg/0.1 ml.

FIGURE 6.

Effect of engagement of α4β1 or α4β7 integrins by mAbs on the survival of eosinophils. Eosinophils were incubated for 3 days with the indicated concentrations of the mAbs Fib 30, TS2/16, or Fab fragments of mAb Fib 30. Data are expressed as the mean ± SEM of four separate experiments for mAb Fib 30 and Fab fragments of Fib 30, and three experiments for TS2/16. Fib 30 was significantly different (p < 0.05) from no mAb addition (0 μg/0.1 ml) at concentrations greater than 0.1 μg/0.1 ml.

Close modal

It is possible that mAb Fib 30 cross-linked α4β7 on eosinophils, sending a signal resulting in extended viability. Eosinophils were incubated with mAbs to β7 (Fib 21, Fib 22, Fib 27, and Fib 30) alone or added with mAb P4C10 to β1 in the absence of cellular fibronectin or other survival stimuli (Table IV). Although addition of anti-β1 mAb P4C10 to anti-β7 mAbs was greater than the no mAb controls, the addition was not significantly more than the mAbs to β7 added alone (Table IV). The inclusion of an anti-mouse mAb to cross-link β1 did not significantly increase viability of eosinophils incubated with anti-β7 mAbs, whether or not P4C10 was present in the assay. Similarly, addition of an anti-rat mAb to cross-link β7 also did not increase eosinophil viability (Table II). Only Fib 30 greatly enhanced eosinophil viability, and it was not significantly increased when used in combination with anti-β1 or anti-rat mAbs (Table IV). Clustering of β7 integrins on the cell surface could have resulted from aggregates of the Fib 30 mAb. However, eosinophils incubated with Fib 30 that was centrifuged to remove aggregates were equally viable as eosinophils incubated with Fib 30 solutions that were not centrifuged (data not shown), indicating that aggregation of the mAb was not the cause of increased survival using this mAb.

Table IV.

Effect of anti-integrin mAbs on eosinophil viabilitya

Rat Anti-β7 mAbsViability of Eosinophils on Day 3 (% of total) at Following Additive Effects of mAbs
No added mAb (6)Anti-rat IgG (2)Mouse anti-β1 mAb P4C10 (5)Anti-mouse IgG (3)Anti-β1 mAb P4C10 + anti-mouse IgG (3)Anti-β1 mAb P4C10 + anti-mouse IgG + anti-rat IgG (2)
None 18 ± 3 28, 16 26 ± 4* 22 ± 2 30 ± 3 ND 
Fib 21 25 ± 4 21b , 43b 37 ± 3*b 28 ± 3b 40 ± 1* 35, 39*b 
Fib 22 27 ± 5 32c , 25c 36 ± 6*c 32 ± 4c 41 ± 3*c 46c , 18c 
Fib 27 25 ± 4 24,d 39d 34 ± 3*d 32 ± 3d 35 ± 2*d 30, 34d 
Fib 30 51 ± 9* 67*, 60*e 61 ± 5*e 61 ± 6*e 63 ± 4*e 63,*e 53*e 
Rat Anti-β7 mAbsViability of Eosinophils on Day 3 (% of total) at Following Additive Effects of mAbs
No added mAb (6)Anti-rat IgG (2)Mouse anti-β1 mAb P4C10 (5)Anti-mouse IgG (3)Anti-β1 mAb P4C10 + anti-mouse IgG (3)Anti-β1 mAb P4C10 + anti-mouse IgG + anti-rat IgG (2)
None 18 ± 3 28, 16 26 ± 4* 22 ± 2 30 ± 3 ND 
Fib 21 25 ± 4 21b , 43b 37 ± 3*b 28 ± 3b 40 ± 1* 35, 39*b 
Fib 22 27 ± 5 32c , 25c 36 ± 6*c 32 ± 4c 41 ± 3*c 46c , 18c 
Fib 27 25 ± 4 24,d 39d 34 ± 3*d 32 ± 3d 35 ± 2*d 30, 34d 
Fib 30 51 ± 9* 67*, 60*e 61 ± 5*e 61 ± 6*e 63 ± 4*e 63,*e 53*e 
a

Data are expressed as the mean ± SEM of three to six experiments, as noted in parentheses, or the values from two experiments.

b

Not significantly different from Fib 21 alone.

c

Not significantly different from Fib 22 alone.

d

Not significantly different from Fib 27 alone.

e

Not significantly different from Fib 30 alone.

f

, p < 0.05 vs no mAb addition.

The panel of mAbs to β7 used in these experiments bind three functional epitopes within aa residues 176–237 (14, 28). Fib 21 and Fib 22 recognize epitope C, and Fib 27 and Fib 30 recognize epitope D1 on β7. The epitope recognized by Fib 504 (D2) appears to overlap both epitopes C and D1, because Fib 504 competes with each of these mAbs in a flow-cytometric competition assay (14). Fab fragments of Fib 504 effectively competed with Fib 30 on human eosinophils, completely blocking survival of eosinophils incubated with Fib 30 (Fig. 7).

FIGURE 7.

Blocking Fib 30 with Fab fragments of Fib 504. Eosinophils were treated for 30 min at 40°C with increasing concentration of Fab fragments of Fib 504 as indicated before incubation with 0.5 μg/0.1 ml anti-β7 mAb Fib 30 (Fib 30 + Fib 504 Fab, ○) or no addition (Fib 504 Fab, ▪). Data are expressed as the mean ± SEM of three experiments. Samples containing Fib 30 + 2.0 μg/0.1 ml Fib 504 Fab were significantly different (p < 0.05) from samples containing Fib 30 + 0 μg/ml Fib 504 Fab.

FIGURE 7.

Blocking Fib 30 with Fab fragments of Fib 504. Eosinophils were treated for 30 min at 40°C with increasing concentration of Fab fragments of Fib 504 as indicated before incubation with 0.5 μg/0.1 ml anti-β7 mAb Fib 30 (Fib 30 + Fib 504 Fab, ○) or no addition (Fib 504 Fab, ▪). Data are expressed as the mean ± SEM of three experiments. Samples containing Fib 30 + 2.0 μg/0.1 ml Fib 504 Fab were significantly different (p < 0.05) from samples containing Fib 30 + 0 μg/ml Fib 504 Fab.

Close modal

It has been reported that survival of eosinophils incubated with coated cellular fibronectin is inhibited by mAbs to GM-CSF, IL-3, and IL-5 (10). We hypothesized that a similar mechanism may account for soluble cellular fibronectin-mediated survival. Eosinophils pretreated with mAbs to IL-3 or IL-5 were equally as viable as eosinophils pretreated with control mouse IgG or eosinophils that were not treated with mAb before incubation with soluble cellular fibronectin (Fig. 8,A). In contrast, treatment with anti-GM-CSF mAb completely blocked cellular fibronectin-mediated survival to levels of samples incubated without cellular fibronectin (Fig. 8,A). This inhibition was not increased when mAbs to IL-3 and/or IL-5 were added with anti-GM-CSF (Fig. 8,A). Similarly, eosinophils were also treated with mAbs to cytokines before incubation with Fib 30 to test whether survival of eosinophils mediated by Fib 30 also was mediated by GM-CSF (Fig. 8,B). Only samples containing the mAb against GM-CSF showed significant decreased viability mediated by Fib 30 (Fig. 8,B), similar to what was observed with survival mediated by soluble cellular fibronectin (Fig. 8 A).

FIGURE 8.

The role of cytokines in enhancement of eosinophil survival by cellular fibronectin or Fib 30. A and B, Eosinophils were treated for 30 min at 40°C with 15 μg/ml mAbs against IL-3, IL-5, GM-CSF, or an isotype-matched control mAb before incubation with medium containing no addition (No cFn or No Fib 30) or medium containing (A) 2.0 μg/0.1 ml cellular fibronectin (hatched bars) or (B) medium containing 0.5 μg/0.1 ml anti-β7 mAb Fib 30 (open bars). Data are expressed as the mean ± SEM of (A) three experiments and (B) four experiments. All samples containing anti-GM-CSF in A and B were significantly different (p < 0.05) from samples containing no mAb. C, Eosinophils (3 × 106) were incubated in medium containing no addition, or medium containing 20 μg/ml cellular fibronectin, 5 μg/ml anti-β7 mAb Fib 30, or control rat IgG, or 10−6 M ionomycin for 6 h. RT-PCR followed by Southern blotting of PCR products was performed as described in Materials and Methods. This experiment was repeated with similar results.

FIGURE 8.

The role of cytokines in enhancement of eosinophil survival by cellular fibronectin or Fib 30. A and B, Eosinophils were treated for 30 min at 40°C with 15 μg/ml mAbs against IL-3, IL-5, GM-CSF, or an isotype-matched control mAb before incubation with medium containing no addition (No cFn or No Fib 30) or medium containing (A) 2.0 μg/0.1 ml cellular fibronectin (hatched bars) or (B) medium containing 0.5 μg/0.1 ml anti-β7 mAb Fib 30 (open bars). Data are expressed as the mean ± SEM of (A) three experiments and (B) four experiments. All samples containing anti-GM-CSF in A and B were significantly different (p < 0.05) from samples containing no mAb. C, Eosinophils (3 × 106) were incubated in medium containing no addition, or medium containing 20 μg/ml cellular fibronectin, 5 μg/ml anti-β7 mAb Fib 30, or control rat IgG, or 10−6 M ionomycin for 6 h. RT-PCR followed by Southern blotting of PCR products was performed as described in Materials and Methods. This experiment was repeated with similar results.

Close modal

Levels of message for GM-CSF were examined in eosinophils incubated with soluble cellular fibronectin or Fib 30 to further investigate this cytokine as a mediator of the survival response to soluble ligands. Eosinophils incubated with soluble cellular fibronectin or Fib 30 contained a greater amount of message for GM-CSF than eosinophils incubated without additions to the medium (Fig. 8,C). Similar to results of Fab fragments of Fib 504 blocking the effect of Fib 30 in the survival assay (Fig. 7), eosinophils pretreated with Fab fragments of Fib 504 before incubation with Fib 30 did not have an increase in message for GM-CSF (data not shown).

In this study, we demonstrate that soluble ligands of α4 integrins increase the survival of eosinophils in culture. The survival response was promoted primarily via α4β7, because mAbs blocking α4β1 only partially inhibited survival mediated by soluble cellular fibronectin and mAb Fib 30 to β7 increased eosinophil viability in the absence of additions ligands. Engagement of α4β7 by soluble ligands (cellular fibronectin or Fib 30) resulted in increased expression of GM-CSF, which extends the viability of eosinophils in culture. This was demonstrated by increased levels of message for GM-CSF in eosinophils incubated with soluble cellular fibronectin or Fib 30 and by the ability of mAbs against GM-CSF to completely inhibit the increased survival of eosinophils incubated with soluble cellular fibronectin or Fib 30.

We found that soluble VCAM-1 and ED-A+ cellular fibronectin, ligands of α4 integrins, induce extended survival of eosinophils in culture, an effect that did not require adherence of eosinophils to these proteins. Soluble cellular fibronectin did not become adherent to the well during the incubation period. The level of detection of the ELISA was 5 ng/ml, which was lower than the lowest concentration of coated cellular fibronectin that yielded a significant level of increased viability (10 μg/0.1 ml coating concentration), indicating that the protein was acting in a soluble form on the eosinophils. Viability was increased whether cellular fibronectin or rsVCAM-1 was coated onto plates or added with the eosinophils in solution at the beginning of the assay. These were unexpected results based on previous studies of fibroblasts, epithelial cells, and endothelial cells. Survival of such cells in culture requires integrin-mediated adhesion (29). When this adhesion is lost, cells die via a specialized form of apoptosis called anoikis (29). In contrast, we found that the matrix protein need not be coated onto the substratum to prevent cell death and that the cells do not need to be adherent to the plate (Fig. 4). The concentrations of rsVCAM-1 and cellular fibronectin that supported increased viability are within the ranges of concentrations estimated to be in airway secretions. Assuming a 50-fold dilution of airway lining fluid by lavage, 25 μg/ml of fibronectin on average (2) and up to 0.6 μg/ml of VCAM-1 (5) are estimated to be present in secretions after Ag challenge. These findings suggest that soluble ligands for α4 integrins may account, at least in part, for the eosinophil survival in the lungs of asthmatics after exposure to Ag and for the strong correlations between BAL VCAM-1 levels and eosinophil levels (5) and between BAL fibronectin levels and eosinophil levels (2).

Eosinophil viability is increased by soluble cytokines, including IL-5 and GM-CSF (30). Such minor contaminants within the protein preparations could alter eosinophil survival. However, we found no IL-5, GM-CSF, RANTES, or IFN-γ in the cellular fibronectin used in these experiments. The active proteins were from different cell sources and purified by different methods, making it further unlikely that the increase in eosinophil viability was due to cytokine contamination. Because the cellular fibronectin and rsVCAM-1 did contain trace endotoxin (up to 1 ng/ml), we conducted the survival assays in the presence of 50 U/ml polymyxin B, a dose previously shown to completely block eosinophil survival induced by 10 ng/ml LPS (Ref. 31 and our unpublished observations). Also, other integrin ligands failed to increase eosinophil viability (plasma fibronectin, type 1 laminin, CS-1 peptides, or soluble ICAM-1), indicating that a specific interaction between cellular fibronectin or VCAM-1 with α4 integrins was responsible for the survival-enhancing effect.

VCAM-1 contains binding sites with the sequence QIDSPL for α4 integrins in the first and fourth Ig-like modules, with synergy sites located in the adjacent modules 2 and 5 (3). The predominant splice variant of VCAM-1 expressed on stimulated endothelium contains both the first and fourth modules (7D-VCAM), although another form can be found that lacks the fourth module (6D-VCAM) (3). The rsVCAM-1 used in these studies contains both the first and fourth modules (16). Fibronectin contains several binding sites for α4 integrins within the C-terminal heparin-binding domain, the variably spliced IIICS or V region (3), and in the fifth type III repeat (32). The higher affinity binding site within the connecting segment-1, LDV (33), is located within the first 25 amino acids of the V region and is present in the 89 amino acid (V89) and 120 amino acid (V120) splice variants secreted by most cultured cells. Thus, the LDV-binding site is present in both subunits of most cellular fibronectin dimers. Splice variants lacking the LDV site constitute at least half of the fibronectin message in hepatocytes responsible for plasma fibronectin (7). Therefore, plasma fibronectin contains only one of the high affinity LDV-binding sites, whereas cellular fibronectin contains two. Cellular fibronectin promoted eosinophil survival whereas plasma fibronectin did not, whether the proteins were coated onto surfaces or used in solution. This is presumably because plasma fibronectin contains one and not two binding sites for α4 integrins. CS-1 peptides did not increase eosinophil survival when added in solution with the cells, again suggesting that the ligand must be in a dimeric form to be active in the survival assay. The possibility that the alternatively spliced ED-A and ED-B modules or the fifth type III repeat are responsible for the increased survival, however, cannot be ruled out. It is clear that the interaction of soluble cellular fibronectin with eosinophils is complex since survival of eosinophils incubated with soluble cellular fibronectin was not completely blocked by pretreatment of mAbs to integrins (data herein) or competed away with excess plasma fibronectin or CS-1 fragments (our unpublished observations).

Since both cellular fibronectin and VCAM-1 contain two binding sites for α4-integrins, it is possible that cross-linking the integrin on the cell surface caused the enhancement of survival. One mAb to β7, Fib 30, increased eosinophil viability without other ligands present (Fig. 6). However, Fab fragments of Fib 30, acting as monomeric ligands, did not increase eosinophil viability (Fig. 6). The mode of action of Fib 30 was not likely to be mediated by interaction through FcγRII (CD32), one of the lower affinity forms of IgG receptors. Fab fragments of Fib 504 competed away the activity of Fib 30, indicating an interference of binding to the ligand, not the Fc receptor. Also, little nonspecific binding of mouse or rat mAbs was detected by flow cytometry (Table II), again suggesting few interactions through Fc receptors. Attempts to cross-link α4 integrins by mAbs resulted in only modest increase in eosinophil viability (Table IV). Only the intact mAb Fib 30 to β7 enhanced eosinophil survival greatly and consistently (Fig. 6, Table IV). Fab fragments of another anti-β7 mAb, Fib 504, effectively blocked eosinophil survival and increased GM-CSF message in eosinophils incubated with Fib 30. Thus, β7 enhanced eosinophil survival through a mechanism involving both the specific epitope recognized by Fib 30 and cross-linking of the integrin as occurs when using intact mAbs or dimeric ligands. It appears both cross-linking and specific interactions with ligand-binding regions contribute to the increase in eosinophil viability.

Increasing evidence suggests that interaction of eosinophils with cell surface adhesion molecules and extracellular matrix proteins alter the cell’s phenotype. Ligation of eosinophil β1 (CD29) or β2 (CD18) integrins by surface-bound anti-integrin mAbs induces cell spreading and triggers the respiratory burst (34). Moreover, eosinophils incubated in fibronectin-coated wells show enhanced degranulation and increased production of leukotriene C4 (35). Eosinophils release increased amounts of superoxide anion when incubated with VCAM-1 in a reaction blocked by anti-α4 mAbs (36). The α4 integrin subunit associates with β1 and β7 subunits on eosinophils, forming two receptors for VCAM-1 and fibronectin (6). On eosinophils, α4β1 and α4β7 integrins require different levels of activation to bind ligands (6), and α4β7 binds to an additional cell surface ligand, mucosal addressin cell adhesion molecule-1 (3, 6). The cytoplasmic tails of β1 and β7 bind to different cytoskeletal and cytoplasmic proteins (37, 38). Both α4β1 and α4β7 have been implicated in eosinophil survival stimulated by coated fibronectin (10, 35). Here, we have shown a role for α4β7 in survival of nonadherent eosinophils. A role for α4β1 may also be important for these interactions since mAbs blocking both α4 and β1 subunits inhibited survival of eosinophils incubated with soluble cellular fibronectin.

A proposed mechanism of eosinophil survival mediated by substrate-bound cellular fibronectin is autocrine generation of GM-CSF and IL-5 (9, 10). Abs to GM-CSF, but not to IL-3 or IL-5, reduced eosinophil viability when cultured in the presence of either cellular fibronectin or Fib 30 (Fig. 8, A and B), suggesting that autocrine generation of GM-CSF is also responsible for eosinophil survival mediated by ligands for α4β7. Indeed, increased levels of message for GM-CSF were found in eosinophils incubated with either soluble cellular fibronectin or Fib 30 (Fig. 8 C). We propose that the GM-CSF protein generated becomes rapidly bound by eosinophils. The amounts generated seem to be very small because they are less than ELISA detection levels (our unpublished observations).

Fibronectin has been found in elevated amounts in lavage fluid of asthmatic patients compared with normal patients, although the type of fibronectin located within the airway space was not determined (39). Cellular fibronectin is found in lavage fluid of rabbits after lung injury mediated by PMA-treated leukocytes or glucose + glucose oxidase to generate H2O2 (40). The increase in cellular fibronectin after Ag challenge may be a result of local inflammatory processes within the airway space. This is supported by the correlation of fibronectin levels and inflammatory leukocytes (including eosinophils) observed in patients given segmental challenge with allergens (2). Segmental allergen challenge also increases the expression of VCAM-1 in lung tissue (41). Increased levels of soluble VCAM-1 have been found in the BAL fluid of allergic and asthmatic patients after Ag challenge (5). The source of VCAM-1 in BAL has not been determined but may be a result of local edema or local production by airway vascular smooth muscle cells (5, 41). The levels of soluble VCAM-1 found in lavage fluid of Ag-challenged subjects also correlates with increased leukocyte numbers in the lavage fluid (5), suggesting that similar mechanisms may be involved in the increased levels of fibronectin and VCAM-1 in lavage fluid.

The increase in eosinophil viability by incubation in wells coated with cellular fibronectin or VCAM-1 suggests a role for these proteins in eosinophil viability in the bronchial wall. The fact that cellular fibronectin and VCAM-1 support eosinophil viability when provided in solution during culture in vitro suggests these ligands, along with cytokines present, may also contribute to the increase in eosinophil number by extending their survival in the airway. Abs to α4β1 block several inflammatory diseases in animal models including contact hypersensitivity and responses to lung Ag challenge (reviewed in Ref. 8). α4β1 may be responsible for leukocyte activation in addition to leukocyte recruitment because, in a sheep model of allergic asthma, mAbs to α4β1 delivered in the lung via inhaled aerosol inhibit late phase response to inhaled allergen without a significant diminution in leukocyte numbers in the lavage fluid (42). Indeed, it may be that interference of interactions with α4 integrin ligands induces cell death, since providing these interactions increases survival. The percentages of apoptotic cells did not change when eosinophils were cultured with cellular fibronectin or rsVCAM-1, even though the amounts of viable cells were increased compared with controls (Fig. 2). Blocking interaction of eosinophils with α4 integrin ligands within the lung tissue could prevent release of eosinophil granule contents and limit cell viability, thus decreasing tissue damage and resulting fibrosis that are hallmarks of chronic asthma. Blocking eosinophil interactions with α4 integrin ligands within the airway space could also limit cell viability and may prevent further hyperresponsiveness, as was demonstrated in the sheep model. These results clearly indicate that the development of peptidomimetic therapies to combat the role of eosinophils in diseases such as asthma requires agents that block both α4β1 and α4β7.

We thank Drs. David Andrew, Hynda Kleinman, Roy R. Lobb, Francisco Sánchez-Madrid, and Robert Rothlein for providing reagents critical to this study. We thank the staff at the Flow Cytometry Facility at the University of Wisconsin Hospital for help with the flow cytometric analysis. We also thank all those who donated blood for these studies and to Janelle Luedke, Lori McAloon, Nicki Theiss, and Heather Gerbyshak for help with eosinophil isolations and Raymond Rodriguez for his expertise with the cytokine ELISAs.

1

This work was supported by individual National Research Service Award HL09519 to J.M. from the National Institutes of Health (NIH) and by institutional Specialized Center of Research Grant HL56396 from NIH.

4

Abbreviations used in this paper: BAL, bronchoalveolar lavage; ED, extra domain; rsVCAM-1, recombinant soluble VCAM-1; EU, endotoxin unit.

1
Arm, J. P., T. H. Lee.
1992
. The pathobiology of bronchial asthma.
Adv. Immunol.
51
:
323
2
Meerschaert, J., E. A. B. Kelly, D. F. Mosher, W. W. Busse, N. N. Jarjour.
1999
. Segmental antigen challenge increases fibronectin in bronchoalveolar lavage fluid.
Am. J. Respir. Crit. Care Med.
159
:
619
3
Carlos, T. M., J. M. Harlan.
1994
. Leukocyte-endothelial adhesion molecules.
Blood
84
:
2068
4
Wardlaw, A. J., F. S. Symon, G. M. Walsh.
1994
. Eosinophil adhesion in allergic inflammation.
J. Allergy Clin. Immunol.
94
:
1163
5
Zangrilli, J. G., J. R. Shaver, R. A. Cirelli, S. K. Cho, C. G. Garlisi, A. Falcone, F. M. Cuss, J. E. Fish, S. P. Peters.
1995
. sVCAM-1 levels after segmental allergen challenge correlate with eosinophil influx, IL-4 and IL-5 production, and the late phase response.
Am. J. Respir. Crit. Care Med.
151
:
1346
6
Walsh, G. M., F. A. Symon, A. I. Lazarovits, A. J. Wardlaw.
1996
. Integrin α4β7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 and fibronectin.
Immunology
89
:
112
7
Hynes, R. O..
1990
. Structure of fibronectins.
Fibronectins
113
Springer-Verlag, New York.
8
Lobb, R. R., M. E. Hemler.
1994
. The pathophysiologic role of α4 integrins in vivo.
J. Clin. Invest.
94
:
1722
9
Anwar, A. R. F., R. Moqbel, G. M. Walsh, A. B. Kay, A. J. Wardlaw.
1993
. Adhesion to fibronectin prolongs eosinophil survival.
J. Exp. Med.
177
:
839
10
Walsh, G. M., F. A. Symon, A. J. Wardlaw.
1995
. Human eosinophils preferentially survive on tissue fibronectin compared with plasma fibronectin.
Clin. Exp. Allergy
25
:
1128
11
Walsh, G. M., A. J. Wardlaw.
1997
. Dexamethosone inhibits prolonged survival and autocrine granulocyte-macrophage colony-stimulating factor production by human eosinophils cultured on laminin or tissue fibronectin.
J. Allergy Clin. Immunol.
100
:
208
12
Walsh, G. M..
1997
. Mechanisms of human eosinophil survival and apoptosis.
Clin. Exp. Allergy
27
:
482
13
Sanchez-Madrid, F., A. M. Krensky, C. F. Ware, E. Strominger, J. L. Robbins, S. J. Burkoff, T. A. Springer.
1982
. Three distinct antigens associated with human T-lymphocyte-mediated cytolysis: LFA-1, LFA-2, and LFA-3.
Proc. Natl. Acad. Sci. USA
79
:
7489
14
Andrew, D. P., C. Berlin, S. Honda, T. Yoshino, A. Hamann, B. Holzmann, P. J. Kilshaw, E. C. Butcher.
1994
. Distinct but overlapping epitopes are involved in α4β7-mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation.
J. Immunol.
153
:
3847
15
Pulido, R., M. J. Elices, M. R. Campanero, L. Osborn, S. Schiffer, Á. García-Pardo, R. Lobb, M. E. Hemler, F. Sánchez-Madrid.
1991
. Functional evidence for three distinct and independently inhibitable adhesion activities mediated by the human integrin VLA-4: correlation with distinct α4 epitopes.
J. Biol. Chem.
266
:
10241
16
Lobb, R., G. Chi-Rosso, D. Leone, M. Rosa, B. Newman, S. Luhowskyj, L. Osborn, S. Schiffer, C. Benjamin, I. Douglas, C. Hession, P. Chow.
1991
. Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1.
Biochem. Biophys. Res. Comm.
178
:
1498
17
Allio, A. E., P. J. McKeown-Longo.
1988
. Extracellular matrix assembly of cell-derived and plasma-derived fibronectins by substrate-attached fibroblasts.
J. Cell. Physiol.
135
:
459
18
Carnemolla, B., L. Borsi, L. Zardi, R. J. Owens, F. E. Barelle.
1987
. Localization of the cellular-fibronectin-specific epitope recognized by the monoclonal antibody IST-9 using fusion proteins expressed in E. coli.
FEBS Lett.
215
:
269
19
Kelly, E. A. B., R. R. Rodriguez, W. W. Busse, N. N. Jarjour.
1997
. The effect of segmental bronchoprovocation with allergen on airway lymphocyte function.
Am. J. Respir. Crit. Care Med.
156
:
1421
20
Hansel, T. T., I. J. M. De Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. Blaser, C. Walker.
1991
. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils.
J. Immunol. Methods
145
:
105
21
Tourkin, A., T. Anderson, E. C. LeRoy, S. Hoffman.
1993
. Eosinophil adhesion and maturation is modulated by laminin.
Cell Adhes. Commun.
1
:
161
22
Jones, K. H., J. A. Senft.
1985
. An improved method to determine cell viability by simultaneous staining with fluorescein diacetate-propidium iodide.
J. Histochem. Cytochem.
33
:
77
23
Altman, S. A., L. Randers, G. Rao.
1993
. Comparison of trypan blue dye exclusion and fluorometric assays for mammalian cell viability determination.
Biotechnol. Prog.
9
:
671
24
Ormerod, M. G., X.-M. Sun, D. Brown, R. T. Snowden, G. M. Cohen.
1993
. Quantification of apoptosis and necrosis by flow cytometry.
Acta Oncologica
32
:
417
25
Gern, J. E., R. Vrtis, E. A. B. Kelly, E. C. Dick, W. W. Busse.
1996
. Rhinovirus produces nonspecific activation of lymphocytes through a monocyte-dependent mechanism.
J. Immunol.
157
:
1605
26
Siegel, A. F., C. J. Morgan.
1996
.
Statistics and Data Analysis
John Wiley & Sons, Inc, New York.
27
Arroyo, A. G., A. Garcia-Pardo, F. Sanchez-Madrid.
1993
. A high affinity conformational state on VLA-4 integrin heterodimers induced by an anti-β 1 chain monoclonal antibody.
J. Biol. Chem.
268
:
9863
28
Tidswell, M., R. Pachynski, S. W. Wu, S.-Q. Qiu, E. Dunham, N. Cochran, M. J. Briskin, P. J. Kilshaw, A. I. Lazarovits, D. P. Andrew, E. C. Butcher, T. A. Yednock, D. J. Erle.
1997
. Structure-function analysis of the integrin β7 subunit: identification of domains involved in adhesion to MAdCAM-1.
J. Immunol.
159
:
1497
29
Meredith, J. E., Jr, M. A. Schwartz.
1997
. Integrins, adhesion and apoptosis.
Trends Cell Biol.
7
:
146
30
Teixeira, M. M., T. J. Williams, P. G. Hellewell.
1995
. Mechanisms and pharmacological manipulation of eosinophil accumulation in vivo.
Trends Pharmacol. Sci.
16
:
418
31
Takanaski, S., R. Nonaka, Z. Xing, P. O’Byrne, J. Dolovich, M. Jordana.
1994
. Interleukin 10 inhibits lipopolysaccharide-induced survival and cytokine production by human peripheral blood eosinophils.
J. Exp. Med.
180
:
711
32
Moyano, J. V., B. Carnemolla, C. Domínguez-Jiménez, M. García-Gila, J. P. Albar, P. Sanchez-Aparicio, A. Leprini, G. Querze, L. Zardi, A. Garcia-Pardo.
1997
. Fibronectin type III5 repeat contains a novel cell adhesion sequence, KLDAPT, which binds activated α4β1 and α4β7 integrins.
J. Biol. Chem.
272
:
24832
33
Komoriya, A., L. J. Green, M. Mervic, S. S. Yamada, K. M. Yamada, M. J. Humphries.
1991
. The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine.
J. Biol. Chem.
266
:
15075
34
Laudanna, C., P. Melotti, C. Bonizzato, G. Piacentini, A. Boner, M. C. Serra, G. Berton.
1993
. Ligation of members of the β1 or the β2 subfamilies of integrins by antibodies triggers eosinophil respiratory burst and spreading.
Immunology
80
:
273
35
Walsh, G. M., A. J. Wardlaw.
1997
. Eosinophil interactions with extracellular matrix proteins: effects on eosinophil function and cytokine production. B. S. Bochner, Jr, ed.
Adhesion Molecules in Allergic Disease
187
Marcel Dekker, Inc, New York.
36
Nagata, M., J. B. Sedgwick, M. E. Bates, H. Kita, W. W. Busse.
1995
. Eosinophil adhesion to vascular cell adhesion molecule-1 activates superoxide anion generation.
J. Immunol.
155
:
2194
37
Pfaff, M., S. Liu, D. J. Erle, M. H. Ginsberg.
1998
. Integrin β cytoplasmic domains differentially bind to cytoskeletal proteins.
J. Biol. Chem.
273
:
6104
38
Rietzler, M., M. Bittner, W. Kolanus, A. Schuster, B. Holzmann.
1998
. The human WD repeat protein WAIT-1 specifically interacts with the cytoplasmic tails of β7-integrins.
J. Biol. Chem.
273
:
27459
39
Mattoli, S., V. L. Mattoso, M. Soloperto, L. Allegra, A. Fasoli.
1991
. Cellular and biochemical characteristics of bronchoalveolar lavage fluid in symptomatic nonallergic asthma.
J. Allergy Clin. Immunol.
87
:
794
40
Peters, J. H., M. H. Ginsberg, C. M. Case, C. G. Cochrane.
1988
. Release of soluble fibronectin containing an extra type III comain (ED1) during acute pulmonary injury mediated by oxidants or leukocytes in vivo.
Am. Rev. Respir. Dis.
138
:
167
41
Georas, S. N., M. C. Liu, W. Newman, L. D. Beall, B. A. Stealey, B. S. Bochner.
1992
. Altered adhesion molecule expression and endothelial cell activation accompany the recruitment of human granulocytes to the lung after segmental antigen challenge.
Am. J. Respir. Cell Mol. Biol.
7
:
261
42
Lobb, R. R., B. Pepinsky, D. R. Leone, W. M. Abraham.
1996
. The role of α4 integrins in lung pathophysiology.
Eur. Respir. J.
9
:
104s