By
From the * Department of Medicine, Department of Pathology, and § Department of Pediatrics,
Harvard Medical School, the
Division of Rheumatology, Immunology, and Allergy, and the
Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts 02115
We evaluated mature peripheral blood eosinophils for their expression of the surface tyrosine
kinase, c-kit, the receptor for the stromal cell-derived cytokine, stem cell factor (SCF). Cytofluorographic analysis revealed that c-kit was expressed on the purified peripheral blood eosinophils from 8 of 8 donors (4 nonatopic and 4 atopic) (mean channel fluorescence intensity 2.0- 3.6-fold, average 2.8 ± 0.6-fold, greater than the negative control). The uniform and selective
expression of c-kit by eosinophils was confirmed by immunohistochemical analysis of peripheral blood buffy coats. The functional integrity of c-kit was demonstrated by the capacity of
100 ng/ml (5 nM) of recombinant human (rh) SCF to increase eosinophil adhesion to 3, 10, and 30 µg/ml of immobilized FN40, a 40-kD chymotryptic fragment of plasma fibronectin, in
15 min by 7.7 ± 1.4-, 5.3 ± 3.3-, and 5.4 ± 0.2-fold, respectively, and their adhesion to 0.1, 0.5, and 1.0 µg/ml vascular cell adhesion molecule-1 (VCAM-1), by 12.7 ± 9.2-, 3.8 ± 2.5-, and
1.7 ± 0.6-fold, respectively. The SCF-stimulated adhesion occurred without concomitant changes
in surface integrin expression, thereby indicating an avidity-based mechanism. rhSCF (100 ng/ml,
5 nM) was comparable to rh eotaxin (200 ng/ml, 24 nM) in stimulating adhesion. Cell adhesion to FN40 was completely inhibited with antibodies against the 4 and
1 integrin subunits,
revealing that the SCF/c-kit adhesion effect was mediated by a single integrin heterodimer,
very late antigen 4 (VLA-4). Thus, SCF represents a newly recognized stromal ligand for the activation of eosinophils for VLA-4-mediated adhesion, which could contribute to the exit of these
cells from the blood, their tissue localization, and their prominence in inflammatory lesions.
Eosinophils are bone marrow-derived granulocytes with
a dominant extravascular distribution primarily in mucosal tissues (1, 2). Eosinophils have been implicated beneficially in host defense against helminthic parasitic infection
(3), in anti-tumor cytotoxicity (7), and in wound healing (10, 11). Conversely, the abundant eosinophils in the
respiratory mucosal tissue from patients with asthma or
rhinitis are believed to contribute to the inflammatory process by releasing preformed, highly cationic granule proteins with cytotoxic effects (12) and by generating lipid mediators, in particular the cysteinyl leukotriene, leukotriene C4,
with attendant vascular and bronchial smooth muscle constrictor action (13). Eosinophils at the foci of tissue inflammation bear membrane markers of activation such as CD69
(14, 15) and exhibit extended survival, which is attributed
to the attenuation of apoptosis by hematopoietic cytokines,
particularly IL-5, and GM-CSF (16, 17).
Integrins, heterodimeric cell surface receptors, participate in the regulation of leukocyte endothelial cell adhesion, transendothelial cell/basement membrane migration,
and localization in inflammatory tissues. Eosinophils express
the very late antigen (VLA)1-4 ( Stem cell factor (SCF, also known as steel factor) is a bone
marrow stromal cytokine central to hematopoiesis (31).
It is also a peripheral tissue product of fibroblasts and endothelial cells (34). SCF exists in two different forms,
soluble and membrane bound, and is the ligand for the c-kit
receptor that is found on primitive hematopoietic cells (38).
Among hematopoietic cells, c-kit is believed to be retained
only by mature tissue mast cells, and thus is a commonly
used marker for the latter (39, 40). Interaction of the c-kit
receptor with SCF stimulates the growth and early differentiation of hematopoietic cells (38) and sustains mast cell
growth and differentiation in cultures of mouse bone marrow (41, 42) and human cord blood (43, 44). In response to cross-linking of the high affinity IgE receptor, Fc We now demonstrate by cytofluorographic and immunohistochemical analyses the surface expression of c-kit receptor
in freshly isolated peripheral blood human eosinophils.
That recombinant human (rh)SCF augments eosinophil adhesion to the VLA-4 ligands, fibronectin, and VCAM-1, establishes the functional integrity of the eosinophil-expressed c-kit.
Thus, SCF represents an abundant stromal ligand with direct
activating effects for human eosinophils as well as mast cells.
Reagents and Antibodies.
rhSCF (catalogue no. 1833-01, lot
no. B6326; Genzyme, Cambridge, MA), rh eotaxin (Endogen,
Inc., Boston, MA), 2 Isolation of Eosinophils from Peripheral Human Blood.
Blood was
collected into sterile, heparinized syringes from the peripheral veins
of nonatopic and atopic volunteer donors who gave informed consent. After the erythrocytes were sedimented with dextran for 45 min at 37°C, the granulocyte fraction was obtained by centrifugation through a cushion of Ficoll-Hypaque (1.77 g/ml; Pharmacia, Uppsala, Sweden) of the buffy coat at 350 g for 30 min. After
the hypotonic lysis of residual erythrocytes, eosinophils were separated from neutrophils by negative immunomagnetic selection with
a magnetic cell separation (MACS) column (Miltenyi Biotec, Sunnyvale, CA) (59). In brief, the erythrocyte-depleted granulocyte
pellet was incubated for 45 min at 4°C with anti-CD16 mAb
bound to immuno-magnetic beads. When the mixture was applied to a steel wire column in a strong magnetic field, the
CD16+ neutrophils were retained, whereas the CD16 Cytofluorographic and Immunohistochemical Analyses of Surface c-kit
Expression on Eosinophils.
Cytofluorographic analyses of surface
epitopes expressed by human peripheral blood eosinophils with or
without rhSCF stimulation were performed by established procedures (52). Freshly isolated human peripheral blood eosinophils were
resuspended in RPMI 1640 medium containing 10% FCS at a
concentration of 106 cells/ml and were divided into two identical
fractions. rhSCF was added to one fraction to a final concentration of 100 ng/ml (5 nM). Alternatively, rh eotaxin was used at a
final concentration of 24 nM and both fractions were incubated
for 15 min at 37°C. The cells were harvested and washed once with
cold PBS containing 0.5% HSA and 0.02% sodium azide (FACS
buffer). Samples of 5 × 105 cells were then incubated for 1 h on ice
with primary antibodies (purified mAbs at a final concentration of
10 µg/ml or ascites at a final dilution of 1:500 or P3 culture supernatant at 1:4 dilution). The cells were washed once with
FACS buffer and incubated in the dark for 1 h on ice with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (GIBCO
BRL) at a final dilution of 1:100. The cells were washed again
with the FACS buffer, resuspended in 0.25 ml of PBS, and analyzed on a FACSort® machine (Becton Dickinson, Oxnard, CA).
For c-kit expression (Fig. 1), the results are presented as overlaid
histograms and the fold increase of mean fluorescence intensity
(MFI). The fold increase of c-kit expression was calculated by dividing the MFI units of SR-1 staining by the MFI units of P3
control mAb staining in each donor.
4
1) and VLA-6 (
6
1) as
well as
4
7 (18). VLA-4 mediates leukocyte attachment to VCAM-1 on activated endothelial cells (18, 21).
Anti-
4 antibodies block eosinophil recruitment and prevent antigen-induced bronchial hyperreactivity in several
animal models, suggesting a critical role for the
4 integrins
in the tissue recruitment, activation, and/or accumulation of eosinophils in allergic disease (22). The VLA-4 integrin also binds to fibronectin through an alternatively
spliced connecting segment-1 (CS-1) region of fibronectin
(27). The interaction between VLA-4 and fibronectin results in prolonged eosinophil survival in culture by inducing the autocrine generation of GM-CSF and IL-3 (28).
Inasmuch as a subpopulation of eosinophils in nasal polyps
(29) and bronchoalveolar lavage fluid from individuals with
asthma undergoing allergen challenge (30) expresses GM-CSF protein and/or mRNA, it is possible that in situ VLA-4-fibronectin interaction prolongs eosinophil retention and
viability through an autocrine mechanism.
R1,
SCF primes mature dispersed human lung mast cells for both
augmented exocytosis of secretory granules (45) and cytokine production (46) and primes mouse bone marrow-
derived mast cells (BMMC) for enhanced generation of
membrane-derived eicosanoids (47). Additionally, SCF is a
direct activator of BMMC, stimulating both exocytosis and
eicosanoid generation with the same biochemical steps and
kinetics as activation by Fc
R1 (48). SCF promotes the adhesion of BMMC to fibronectin via
1 integrins, increasing the
5
1 (VLA-5) integrin avidity (49). Thus, SCF is a
potentially critical regulatory factor in the localization, proliferation, priming, and direct activation of mast cells.
,7
-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) (Molecular Probes,
Eugene, OR), human serum albumin (HSA) (Sigma Chem. Co.,
St. Louis, MO), purified anti-human c-kit YB5.B8 (PharMingen,
San Diego, CA), purified anti-human c-kit (95C3) (Coulter, Miami, FL), purified anti-human integrin
1 (CD29) (4B4) (Coulter), purified anti-human integrin
4 (CD49d) (A4-PUJ1) (Upstate Biotechnology, Lake Placid, NY), purified anti-human CD3
(HIT3a) (PharMingen), mouse laminin and the 40-kD chymotryptic fragment of human fibronectin (FN40) (GIBCO BRL, Gaithersburg, MD) were purchased. The preparation of purified VCAM-(mouse C
) fusion protein (VCAM-1-
) was previously
reported (52). Anti-human integrin
4 (CD49d) (B5G10, ascites)
(53), anti-human integrin
6 (CD49f) (450-33D, ascites) (54),
anti-human integrin
1 (CD29) (A-1A5, ascites) (55), purified anti-human integrin
4
7 (Act-1) (56), and the negative control antibody P3 (hybridoma culture supernatant) (57) were provided by
Dr. M.E. Hemler (Dana-Farber Cancer Institute, Harvard Medical School). Purified anti-human c-kit mAb SR-1 (ascites) (58) was a
gift from Dr. V. Broudy (University of Washington, Seattle, WA).
eosinophils were highly purified in the fraction that flowed through the
column. Contaminating T lymphocytes and monocytes were further removed by incubating the CD16
fraction for another 15 min at 4°C with saturating concentrations of anti-CD3 and anti-CD14 magnetic beads, respectively (Miltenyi). Cytocentrifugation slides of the eosinophils stained with Wright's and Giemsa
stains showed that the purity of the isolated eosinophils was
greater than 95% in all experiments.
Fig. 1.
Cytofluorographic analysis of c-kit receptor expression on
freshly isolated, human peripheral blood eosinophils. (A) Eosinophils from
donors 1 and 2 were analyzed with three different mouse anti-human c-kit
mAbs, SR-1 (), YB5.B8 (----), and 95C3 (.....), as well as a control
mouse mAb, P3 (
), (IgG control). (B) Eosinophils from an additional
6 donors (3) were analyzed with SR-1 and P3 mAbs. The values expressed on the y axis are values of the MFI units of SR-1 staining divided
by the MFI units of P3 control mAb staining in each donor.
[View Larger Version of this Image (35K GIF file)]
Cell Adhesion Assay.
Cells were attached in stasis to FN40,
VCAM-1, and laminin as described (52). FN40 and laminin at concentrations of 3 µg/ml, 10 µg/ml, and 30 µg/ml were coated
onto non-tissue culture grade 96-well microtiter plates (Nunc-Immuno Plate) in 0.1 M NaHCO3 (pH 8.3) (100 µl/well) for 16 h
at 4°C. The plates were washed twice with PBS (150 µl/well),
and the nonspecific binding sites were blocked by incubation
with 5% HSA in PBS (100 µl/well) for 45 min at 37°C. After
two washes with PBS (150 µl/well), the plates were ready to be
used. For the VCAM-1 assay, plates were precoated with goat
anti-mouse (GIBCO BRL) at a 1:1,000 final dilution in 0.1 M
NaHCO3 (100 µl/well) for 16 h at 4°C. After two washes with
PBS, the plates were coated with VCAM-1-
at concentrations of 0.1, 0.5, and 1.0 µg/ml in 0.1 M NaHCO3 (100 µl/well) for
2 h at 4°C. The plates were washed with PBS and blocked with
HSA as mentioned above. The freshly isolated eosinophils were
incubated with BCECF-AM (5 µg/ml) in RPMI 1640 containing
10 mM Hepes and 0.1% HSA (RPMI-HSA) for 30 min at 37°C,
washed twice with PBS, and resuspended in RPMI-HSA at 4 × 106 cells/ml. Samples (50 µl) of the cell suspension containing
2 × 105 cells were added to each well of the ligand-coated plate;
each well had been preloaded with either 50 µl of RPMI-HSA or
50 µl of RPMI-HSA containing defined concentrations of
rhSCF (generally 10 nM) or defined concentrations of rh eotaxin
(generally 48 nM). The plates were incubated for 15 min at 37°C,
and the fluorescence of total input cells was quantitated by a fluorescence analyzer (Idexx Laboratories, Westbrook, ME). Unbound cells were removed by washing the plates with RPMI/10
mM Hepes 4-5 times at 150 µl/well until the cells in the control
wells (5% HSA alone) were less than 5% of their total input. Cells
remaining attached to the plate were quantitated after every wash
with the same fluorescence analyzer. The percentage of specific
binding to immobilized integrin ligands was calculated as the fluorescence of the cells detected after the final wash divided by the
fluorescence of the total starting cells × 100, and assay results are
presented as means ± SD of three independent experiments, each
performed in triplicate.
Statistical Analysis. The statistical significance of differences between sample means for each set of cells was based on comparison as determined by the Student's t test for matched pairs. Results are presented as mean ± SD.
To determine whether freshly isolated, peripheral human blood eosinophils expressed the c-kit receptor, the purified cells from eight separate donors (four nonatopic and four atopic) were analyzed by cytofluorography for the expression of c-kit. Three mouse mAbs against three independent epitopes of the human c-kit (58, 60) gave virtually identical expression for two of the donors (Fig. 1 A), with mean log fluorescence intensities of 2.0- and 3.3-fold over control, respectively, with mAb SR-1. The surface c-kit expression was subsequently confirmed in six additional donors with one of the three mAbs, SR-1 (Fig. 1 B). In every case, the c-kit expression was readily detectable, with a mean log fluorescence intensity of 2.0 to 3.6-fold greater than the negative control (IgG control mAb P3) (mean 2.8 ± 0.6 fold, n = 8).
To confirm c-kit expression by peripheral blood eosinophils and determine its potential expression by other circulating leukocytes, peripheral blood buffy coats from two
separate donors (1 atopic and 1 nonatopic) were incubated
with either SR-1 or control P3 antibody, and were subjected to immunohistochemical analysis using secondary
antibody-conjugated gold particles and a silver enhancement procedure, followed by counterstaining with hematoxylin and eosin. As indicated by the counterstaining and
shown for the nonatopic donor (Fig. 2), 100% of the eosinophils in the buffy coats were positive for c-kit, and eosinophils were the only cells displaying a signal for c-kit receptor. Identical results were obtained for the atopic donor
(data not shown). A similar positive signal was also detected
on freshly isolated human peripheral blood eosinophils after
MACS column purification (data not shown).
Effect of rhSCF on Eosinophil Adhesion to FN40 and rVCAM-1.
To determine the functional integrity of the
expressed c-kit receptor on eosinophils, the ability of
rhSCF to augment their adhesion to ligands selective for
integrin 4 and
6 was evaluated in a static adhesion assay.
The 15-min time course for the adhesion assay was selected
because a kinetic study showed that augmented adhesion
peaked at 15 min, persisted for 30 min, and decreased to baseline by 45 min (data not shown). Adhesion to FN40 increased
at all three inputs of 3, 10, and 30 µg/ml of immobilized
FN40. In the absence of rhSCF, the adhesion of FN40 was
limited (specific binding of 4.3 ± 3.5% at 30 µg/ml
FN40). Concomitant stimulation with rhSCF (5 nM) augmented adhesion to 3, 10, and 30 µg/ml FN40 by 7.7 ± 1.4-fold, 5.3 ± 3.3-fold, and 5.4 ± 0.2-fold, respectively,
compared with baseline (Fig. 3). Adhesion to rVCAM-1 also
increased in relation to the input of ligand. In the absence
of rhSCF, rVCAM-1 supported greater adhesion than
FN40 (specific binding of 9.3 ± 9% and 22.5 ± 10% at 0.5 µg/ml and 1.0 µg/ml of rVCAM-1, respectively). Stimulation with rhSCF augmented adhesion to 0.1, 0.5, and 1.0 µg/ml rVCAM-1 by 12.7 ± 9.2-fold, 3.8 ± 2.5-fold, and
1.7 ± 0.6-fold, respectively, compared with baseline (Fig. 3).
Higher baseline adhesion mediated by VCAM-1 compared
with fibronectin has been previously reported (22). Cell adhesion to laminin also increased in a dose-dependent fashion with respect to ligand input but did not increase further with stimulation by rhSCF (Fig. 3). Treatment with rhSCF
did not change the surface expression of integrin
4,
6,
1,
4
7, or c-kit receptor as evaluated by cytofluorographic analysis (Fig. 4), indicating that the increases in adhesion to the
4 ligands were not due to increased receptor expression.
Effect of Antibody Neutralization on rhSCF-stimulated Adhesion of Eosinophils to FN40.
Both of the 4 integrins expressed on eosinophils, VLA-4 (
4
1) and
4
7, serve as receptors for FN40 and VCAM-1 (19). Antibody-blocking experiments were performed in the assay for adhesion of
eosinophils to 30 µg/ml FN40 with or without rhSCF
stimulation (5 nM). Although the fold increase in response
to rhSCF was similar at each concentration of FN40 (see
Fig. 3), the greatest absolute increment in adhesion occurred at 30 µg/ml FN40. mAbs against
4 (A4-PUJ1) and
1 (4B4) completely abolished eosinophil adherence to FN40,
whereas the anti-
4
7 mAb (Act-1) had no effect and was
comparable to the control mAb of irrelevant specificity
(anti-CD3) (Fig. 5). Similarly, cell adhesion to rVCAM-1
in the absence or presence of rhSCF (5 nM) was also completely blocked by anti-
4 and anti-
1 mAbs (data not
shown). Therefore, rhSCF-c-kit ligation specifically augmented the adhesion of VLA-4 to its ligands, fibronectin, and VCAM-1.
Dose Effect of rhSCF on Eosinophil Adhesion to FN40.
The effect of rhSCF at concentrations ranging from 12.5 ng/ml (0.625 nM) to 200 ng/ml (10 nM) on eosinophil adhesion to 30 µg/ml FN40 was studied. Enhancement of
VLA-4 binding to FN40 by rhSCF was significant at 2.5, 5, and 10 nM as compared with the unstimulated replicate
eosinophils (P <0.05) (Fig. 6). Each fourfold increment in
rhSCF concentration produced a statistically significant gain in binding (P <0.05) except for the final increment
between 2.5 and 10 nM, suggesting that a plateau was
reached.
Comparison of the Effects of rhSCF and rh Eotaxin on Eosinophil Binding to FN40.
Eotaxin, a selective eosinophil
chemoattractant (61, 62), belongs to the CC chemokine
family. Other members of this family are known to be
stimuli for eosinophil adhesion via integrin 4 (63). Preliminary dose-dependence experiments indicated that the effect of 24 nM rh eotaxin was similar to the effect of 5 nM
rhSCF for augmenting eosinophil adhesion to 30 µg/ml
FN40. Thus, the effects of these concentrations on eosinophil adhesion were compared over three concentrations of
FN40 (Fig. 7). Eotaxin at 24 nM increased eosinophil binding to FN40 to the same extent as 100 ng/ml (5 nM) rhSCF
at each static input of FN40 ligand. Eotaxin did not change
the surface expression of c-kit,
4,
6,
1, and
4
7 by
cytofluorographic analysis (data not shown), and the augmented adhesion of these cells to FN40 produced by eotaxin was completely blocked by mAbs against
4 and
1
(Fig. 8).
The dose-related effect of rh eotaxin on human eosinophil adhesion to FN40 (30 µg/ml) was analyzed in the absence and presence of 1.25 nM rhSCF. The approximate
EC50 for rh eotaxin-augmented adhesion occurred at the
lowest concentration studied, 3 nM, and the plateau was
reached at 12 nM (20.8 ± 13-fold, n = 3). The effect of
the concomitant presence of rhSCF at slightly less than its
EC50 (see Fig. 6) was generally somewhat additive and did not extend the maximum reached with the plateau doses of
rh eotaxin alone (Fig. 9).
The finding that mature human peripheral blood eosinophils express a functional c-kit receptor for SCF reveals that a stromal ligand can activate effector cells conventionally linked to allergic inflammation. Although originally appreciated for its central role in the hematopoiesis of all lineages, SCF is elaborated in the peripheral microenvironment by diverse cell types, including endothelial cells and fibroblasts (34). In addition to the functional implications for eosinophil- and mast cell-directed inflammation, the study also uncovers a potential limitation to the use of c-kit detection as a marker of mature mast cells.
In the initial experiments, the surface expression of the c-kit receptor was established by cytofluorographic analysis of freshly isolated human peripheral blood eosinophils. The expression was similar among the cells of all eight donors tested (Fig. 1), irrespective of the presence of donor atopy by history. To exclude an unsuspected specificity, three mAbs directed against separate epitopes of the human c-kit were shown to yield nearly identical fluorescence signals. The positive surface c-kit expression was also demonstrated by immunohistochemical analysis of peripheral blood buffy coats (Fig. 2). Eosinophils were uniformly positive for c-kit expression and were the only cell type expressing c-kit in these preparations. The presence of c-kit was then confirmed by the functional, dose-related response of the eosinophils to SCF signal in static adhesion assays (Fig. 6). An earlier study did not detect a c-kit signal on peripheral blood eosinophils, and functional assays with SCF were not performed (64). However, this same study did demonstrate low level c-kit expression by human peripheral blood basophils and a priming effect of SCF on their IgE-dependent release of histamine (64). Because eosinophils and basophils are closely related and arise from a common progenitor (65), the expression of c-kit and their activation via SCF can now be added to the list of their shared characteristics. Moreover, a triad of hematopoietic allergic effector cells, mast cells, basophils, and eosinophils, would appear to share the expression of c-kit.
rhSCF-c-kit stimulated eosinophil adhesion to FN40
and VCAM-1, but not to laminin (Fig. 3), implying a response via an 4 integrin. Antibody-blocking studies revealed that the adhesion of eosinophils to FN40 (Fig. 5)
and VCAM-1 (data not shown) was mediated by VLA-4
without the involvement of
4
7. Because the surface integrin receptor numbers were unchanged after treatment of
the cells with rhSCF, including
4,
6,
1, and
4
7 (Fig.
4), the enhanced adhesiveness of VLA-4 induced by rhSCF was likely due to increased avidity of VLA-4 to its ligands.
This observation is supported by an earlier study with the
transformed human cell line MO7E (66). The
4
7 integrin did not contribute to the fibronectin and VCAM-1
binding, suggesting that the avidity change of VLA-4 by
SCF may be conveyed through either the unique
subunit
or the combined effect on the
and
heterodimer. The
fact that rhSCF did not increase VLA-6 (
6
1)-mediated
adhesion to laminin favors the latter hypothesis. Thus, the
SCF-c-kit engagement on human peripheral blood eosinophils provides an inside-out signal to increase transiently
the avidity of an integrin, VLA-4, for its cell surface and
matrix ligands.
Recently, Weber and colleagues (63) demonstrated that
the CC chemokines RANTES and MCP-3, as well as the
anaphylatoxin C5a, rapidly increased 4 integrin avidity on
eosinophils and augmented their adhesion to VCAM-1 and
fibronectin with a peak at 15 min after stimulation. This result prompted us to examine the effect of another CC
chemokine, eotaxin, on eosinophil adhesive function. The
EC50 values of 1.25-2.5 nM for rhSCF (Fig. 6) and of 3 nM for rh eotaxin (Fig. 9) were similar with 30 µg/ml of
FN40. Similarly, the magnitudes of eosinophil binding to
the inputs of 3, 10, and 30 µg/ml of FN40 were similar at
the plateau concentrations of rhSCF and of rh eotaxin (Fig. 7). The cell adhesion response to each stimulus (rhSCF and
rh eotaxin) for the ligand FN40 depended entirely on VLA-4,
as shown by mAb inhibition (Figs. 5 and 8). Stimulation of
the peripheral blood eosinophils with less than the EC50 for
the stromal cytokine, rhSCF, and incremental amounts of
the chemokine, rh eotaxin, appeared additive, and the combination neither increased the maximal cellular response nor
revealed a negative counterregulatory action for the common target, VLA-4.
Cell migration is a complex process requiring constant
changes of the cell-substratum bonds with formation of new
bonds (adhesion) at the leading edge of a cell and breakdown of old bonds (deadhesion) at the rear of the cell (67).
This transient response is essential for adhesion to be followed by random or directed cell migration. Progressive
movement within a tissue may require integrated activation of one integrin and resultant negative regulation of another
integrin on the same cell as the ligand binding requirements
change. For example, in Chinese hamster ovary cells, engagement of the transfected human IIb
3 with its specific
ligand caused a transdominant inhibition of other integrins
and resulted in suppressed adhesion of endogenous hamster
5
1 to fibronectin, as well as transfected human
2
1 to
collagen (68). In another study, the activation of the transfected integrin
IIb
3 in Chinese hamster ovary cells, detected
by mAb PAC1 binding, was suppressed by the introduction
of H-Ras and Raf-1 to these cells (69). This suppressive activity was correlated with activation of the ERK MAP kinase pathway and did not involve other dual specificity kinase
sequences such as SEK-c-Jun or MKK-p38 MAP kinase
(69). Thus, the activation of a particular integrin by cytokines such as SCF, which leads to an enhanced binding of
that integrin to its ligand, may contribute to an integrated
signal for coordinated cellular events such as cell migration.
Although found in small numbers in the circulation of healthy people, eosinophils mainly reside in the mucosal tissues in vivo, with an estimated tissue:circulation ratio of 200:1 (1). The mechanisms involving the tissue-specific localization of eosinophils are not commonly addressed, as most studies have focused on the incremental recruitment with immunologically elicited inflammation. SCF is a normal constitutive product of endothelial cells and fibroblasts and can be detected in the serum (36, 70). Therefore, it is a candidate to mediate a basal level interaction between circulating eosinophils and endothelial cells by facilitating interaction between VLA-4 and VCAM-1 at very low levels of VCAM-1 expression (71). Alternatively, exposure of eosinophils to SCF in the extracellular matrix may mediate their transient attachment to fibronectin, altering their migration speed and influencing their tissue localization. Indeed, Palecek and colleagues (72) have demonstrated that at low concentrations of ligand, cell migration speed increased as integrin binding affinity increased. Thus, SCF is a candidate for participating in the tissue distribution of eosinophils under normal physiologic conditions.
The interaction of VLA-4 and VCAM-1 plays a key role
in lymphocyte and eosinophil adhesion and extravasation in
both in vitro and in vivo models of allergic inflammation
(18, 22). VCAM-1 expression is upregulated in the
eosinophil-rich, inflamed airway tissue of individuals with
asthma (73, 74). VLA-4 is expressed on eosinophils and
lymphocytes, but not neutrophils; and the c-kit receptor
expression is limited to eosinophils among these three cell
types. Thus, under conditions of allergic inflammation, local SCF could contribute both by priming mast cells to
elaborate IL-4 (46, 75) and TNF- (75) with consequent
upregulation of endothelial cell VCAM-1 expression (76),
and by mediating a transiently increased avidity for that
ligand by eosinophil VLA-4. This possibility is supported
by a recent finding in mice of the association of allergen
challenge-induced eosinophilic airways inflammation with
increased levels of histamine and SCF in bronchoalveolar lavage fluid and of SCF in serum (70). In that study, the administration of antibody to SCF before allergen challenge
markedly decreased histamine release and pulmonary eosinophil infiltration. The inflammatory response induced by
allergen challenge also increases the production of the eosinophil-selective chemoattractant, eotaxin (61), which is a product of a number of cell types including epithelial cells, endothelial cells, fibroblasts, and even eosinophils (62). Therefore, increased concentrations of eotaxin and SCF could provide
a coordinated signal for the selective recruitment of eosinophils, beginning with transiently enhanced eosinophil adhesion to endothelial cells within the vasculature through
VLA-4-VCAM-1 interaction, followed by directed tissue
movement through adhesion/deadhesion in a chemotactic gradient, and final eosinophil tissue retention through regulated integrin-matrix interaction. Such a matrix interaction
may also prolong eosinophil survival through autocrine production of IL-3 and GM-CSF initiated by VLA-4-fibronectin interaction (28).
We have provided evidence that SCF is an agonist for eosinophil adhesion. These findings have potential relevance for physiologic eosinophil trafficking and for allergic inflammation, in which mast cell activation and eosinophil accumulation are both prominent features.
Address correspondence to Qian Yuan, M.D., Ph.D., Harvard Medical School, Seeley G. Mudd Building, Room 618, 250 Longwood Avenue, Boston, MA 02115. Phone: 617-432-1995; FAX: 617-432-0979.
Received for publication 3 April 1997 and in revised form 13 May 1997.
1Abbreviations used in this paper: BCECF-AM, 2This work was supported by National Institutes of Health grants AI-22531, AI-31599, AI-01304, and HL-36110. Q. Yuan is the recipient of a research fellowship from the Charles A. King Trust and The Medical Foundation.
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