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4ß7 Integrins by Monoclonal Antibodies or Ligands Enhances Survival of Human Eosinophils In Vitro1
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Department of Medicine, University of Wisconsin, Madison, WI 53706; and
Department of Biological Sciences, St. Cloud State University, St. Cloud, MN 56301
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrins, are enriched in the fluid of
airways of allergic patients subjected to Ag challenge. We therefore
hypothesized that ligands of 4 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
ß7, but not mAb P4C10 or TS2/16 to ß1,
increased eosinophil survival. The increased survival of eosinophils
incubated with Fib 30 was blocked by Fab fragments of another
anti-ß7 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 4ß7 by soluble ligand
(or mAb) that stimulates autocrine production of GM-CSF and promotes
eosinophil survival. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Eosinophils undergo diapedesis and interact with their environment via
cell surface integrins (4). Integrins are heterodimers of
and ß subunits that noncovalently associate to form receptors for
extracellular matrix proteins and certain cell surface ligands
(3). Eosinophils express the integrin
4ß1 (very late Ag-4;
CD49d/CD29), which binds VCAM-1 and fibronectin (3).
The 4 integrin subunit also associates
with the ß7 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 4 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
4 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 4ß1 in
several inflammatory diseases, including contact hypersensitivity and
responses to lung Ag challenge in animal models, using
function-blocking Abs against
4ß1
(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
4 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 4ß7 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
4ß7 via autocrine
production of GM-CSF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
mAbs and integrin ligands
Hybridoma cell lines producing mAbs to
ß1 (TS2/16, Ref. 13), to
ß7 (Fib 504 and Fib 21, Ref. 14),
and to ß2 (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 ß7
(Fib 21, Fib 22, Fib 27, and Fib 30) were a generous gift from David P.
Andrew (Amgen, Boulder, CO). mAbs P4C10 to ß1
and P4C2 to 4, 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 4 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-
, 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.
Eosinophil isolation
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.
Eosinophil culture
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 3x with HBSS, and purified eosinophils (1 to 2 x 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.
Determination of eosinophil viability
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 x 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).
Analysis of integrins on eosinophils by flow cytometry
Eosinophils (6 x 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).
Analysis of GM-CSF message in eosinophils
Eosinophils (3 x 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.
Statistics
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrins on viability
of eosinophils in culture
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
4 integrins, and found that wells coated with
at least 1.0 µg/0.1 ml rsVCAM-1 increased eosinophil viability (Fig. 1A).
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). 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. 1B). 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. 1B), even at concentrations as high
as 20 µg/0.1 ml (data not shown). Peptides containing the LDV high
affinity binding site for 4 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. 1B), although with less of an effect than when coated onto
the bottom of the well. Type 1 laminin, a ligand for
6ß1, 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
ß2 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).
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). 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).
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). 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).
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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. 4A, 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. 4A, Nonadherent). A higher
proportion of the nonadherent cells was viable than the adherent cells
(Fig. 4B), indicating that prolonged adhesion of the
eosinophils to the bottom or sides of the wells was not necessary.
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4 integrins on survival of
eosinophils incubated with soluble cellular fibronectin
Flow cytometry was used to assess expression levels of integrins
on eosinophils and also to compare the relative affinities of the rat
anti-mouse ß7 mAbs (Table II). Eosinophils stained strongly with
anti-ß2 mAb TS1/18 (Table II). The level of
ß1 staining with P4C10 was slightly lower than
staining with anti-4 mAb HP2/4.
Eosinophils also stain positively with the
ß1-activating mAb TS2/16 (Table II). The
anti-ß7 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-ß7 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
ß7 integrins (14), and so was used
as a control in survival experiments.
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4 or P4C10 to ß1
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 4 and
ß1 (Table III, 28% average inhibition,
n = 2). Because mAbs to
4ß1 did not completely
block survival of eosinophils incubated with soluble cellular
fibronectin, a panel of mAbs to ß7 were tested
for their ability to decrease eosinophil viability (Fig. 5). Anti-ß7 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 ß7 increased eosinophil survival in
the presence of soluble cellular fibronectin (Fig. 5).
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4ß1 or
4ß7 by mAbs on the viability of
eosinophils in culture
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
ß7 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-ß7 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 ß1 integrin into an active
structural conformation (27), also did not increase
eosinophil viability (Fig. 6). In one experiment, the anti-ß2
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 4 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).
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4ß7 on eosinophils,
sending a signal resulting in extended viability. Eosinophils were
incubated with mAbs to ß7 (Fib 21, Fib 22, Fib
27, and Fib 30) alone or added with mAb P4C10 to
ß1 in the absence of cellular fibronectin or
other survival stimuli (Table IV).
Although addition of anti-ß1 mAb P4C10 to
anti-ß7 mAbs was greater than the no mAb
controls, the addition was not significantly more than the mAbs to
ß7 added alone (Table IV). The inclusion of an
anti-mouse mAb to cross-link ß1 did not
significantly increase viability of eosinophils incubated with
anti-ß7 mAbs, whether or not P4C10 was
present in the assay. Similarly, addition of an anti-rat mAb to
cross-link ß7 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-ß1 or anti-rat
mAbs (Table IV). Clustering of ß7 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.
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).
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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. 8A). In
contrast, treatment with anti-GM-CSF mAb completely blocked
cellular fibronectin-mediated survival to levels of samples incubated
without cellular fibronectin (Fig. 8A). This inhibition was
not increased when mAbs to IL-3 and/or IL-5 were added with
anti-GM-CSF (Fig. 8A). 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. 8B). Only samples containing the mAb against
GM-CSF showed significant decreased viability mediated by Fib 30 (Fig. 8B), similar to what was observed with survival mediated by
soluble cellular fibronectin (Fig. 8A).
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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). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 integrins increase the survival of
eosinophils in culture. The survival response was promoted primarily
via 4ß7, because mAbs
blocking 4ß1 only
partially inhibited survival mediated by soluble cellular fibronectin
and mAb Fib 30 to ß7 increased eosinophil
viability in the absence of additions ligands. Engagement of
4ß7 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 4 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 4
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- 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
4 integrins was responsible for the
survival-enhancing effect.
VCAM-1 contains binding sites with the sequence QIDSPL for
4 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
4 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 4 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 4-integrins, it is possible that
cross-linking the integrin on the cell surface caused the enhancement
of survival. One mAb to ß7, 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 FcRII (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 4 integrins by mAbs resulted in
only modest increase in eosinophil viability (Table IV). Only the
intact mAb Fib 30 to ß7 enhanced eosinophil
survival greatly and consistently (Fig. 6, Table IV). Fab fragments of
another anti-ß7 mAb, Fib 504, effectively
blocked eosinophil survival and increased GM-CSF message in
eosinophils incubated with Fib 30. Thus, ß7
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 ß1
(CD29) or ß2 (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-4 mAbs (36). The
4 integrin subunit associates with
ß1 and ß7 subunits on
eosinophils, forming two receptors for VCAM-1 and fibronectin
(6). On eosinophils,
4ß1 and
4ß7 integrins require
different levels of activation to bind ligands (6), and
4ß7 binds to an
additional cell surface ligand, mucosal addressin cell adhesion
molecule-1 (3, 6). The cytoplasmic tails of
ß1 and ß7 bind to
different cytoskeletal and cytoplasmic proteins (37, 38).
Both 4ß1 and
4ß7 have been
implicated in eosinophil survival stimulated by coated fibronectin
(10, 35). Here, we have shown a role for
4ß7 in survival of
nonadherent eosinophils. A role for
4ß1 may also be
important for these interactions since mAbs blocking both
4 and ß1 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
4ß7. Indeed, increased
levels of message for GM-CSF were found in eosinophils incubated with
either soluble cellular fibronectin or Fib 30 (Fig. 8C). 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
4ß1 block several
inflammatory diseases in animal models including contact
hypersensitivity and responses to lung Ag challenge (reviewed in Ref.
8). 4ß1
may be responsible for leukocyte activation in addition to leukocyte
recruitment because, in a sheep model of allergic asthma, mAbs to
4ß1 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 4 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 4
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
4 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
4ß1 and
4ß7.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. JoAnn Meerschaert, Department of Biological Sciences, St. Cloud State University, 220 Mathematics and Science Center, 720 Fourth Avenue South, St. Cloud, MN 56301-4498. E-mail address:
3 Current address: First Department of Internal Medicine, Showa University School of Medicine, Tokyo, Japan.
4 Abbreviations used in this paper: BAL, bronchoalveolar lavage; ED, extra domain; rsVCAM-1, recombinant soluble VCAM-1; EU, endotoxin unit.
Received for publication June 2, 1999. Accepted for publication September 20, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4ß7 mediates human eosinophil interaction with MAdCAM-1, VCAM-1 and fibronectin. Immunology 89:112.[Medline]
4 integrins in vivo. J. Clin. Invest. 94:1722.
4ß7-mediated adhesion to vascular cell adhesion molecule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation. J. Immunol. 153:3847.[Abstract]
4 epitopes. J. Biol. Chem. 266:10241.4ß1 and 4ß7 integrins. J. Biol. Chem. 272:24832.4 integrins in lung pathophysiology. Eur. Respir. J. 9:104s.
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), 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.
), 2.0 µg/0.1 ml cellular
fibronectin (cFn, 
). 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).
). 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.