By
From * The Johns Hopkins University School of Medicine, Division of Clinical Immunology,
Baltimore, Maryland 21224; and ICOS Corporation, Bothell, Washington 98021
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Abstract |
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The 2 family of integrins, CD11a, CD11b, CD11c, and
d, are expressed on most leukocytes. We show that the newest member of this family,
d, is expressed on human eosinophils
in peripheral blood, and surface expression can be upregulated within minutes by phorbol ester
or calcium ionophore A23187. Culture of eosinophils with interleukin 5 (IL-5) leads to a two-
to fourfold increase in
d levels by 3-7 d without a change in
4 integrin expression. Eosinophils isolated from late phase bronchoalveolar lavage fluids express
d at levels similar to that
seen after 3 d of IL-5 culture. Regarding
d
2 ligands, in both freshly isolated and IL-5-cultured eosinophils, as well as
d
2-transfected Chinese hamster ovary cells,
d
2 can function
as a ligand for vascular cell adhesion molecule 1 (VCAM-1). This conclusion is based on the
ability of monoclonal antibodies to
d,
2, or VCAM-1 to block cell attachment in static adhesion assays. In experiments with eosinophils, the relative contribution of
d
2 integrin-
mediated adhesion is enhanced after IL-5 culture. These experiments demonstrate that
d
2 is an alternative ligand for VCAM-1, and this integrin may play a role in eosinophil adhesion to
VCAM-1 in states of chronic inflammation.
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Introduction |
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Eosinophils have been shown to play an important role in a variety of inflammatory diseases (1). Besides their postulated importance in parasitic infections, these cells are felt to participate in the pathogenesis of allergic disease. In asthma, for example, eosinophils are selectively recruited into the lung, where release of their products, such as granule proteins and leukotrienes, contributes to the airway damage seen in asthma (2, 3). Indeed, one of the possible mechanisms by which corticosteroids work in asthma is that they substantially decrease eosinophil numbers both in the lung and peripheral circulation (4, 5).
Integrins are a class of heterodimeric surface molecules
involved in cellular adhesion (6). They are expressed on
leukocytes and other cells, and are composed of both an and a
chain. Based on shared
subunits these molecules
can be classified into families. Eosinophils express members
of the
1,
2, and
7 integrin families, and in many respects their integrin expression resembles that of other leukocytes (7). However, because human eosinophils express
4
1 and
4
7 integrins, but normal human neutrophils do not, their interaction with one of their ligands, vascular cell adhesion molecule 1 (VCAM-1), is felt to be a mechanism by which selective recruitment of eosinophils into
sites of allergic inflammation occurs (8).
Recently a fourth 2 integrin,
d
2, was identified
and found to be most homologous to CD11b/CD18 and
CD11c/CD18 (11).
d
2 is expressed on most human
leukocytes, including neutrophils, monocytes, and, to a
lesser extent, lymphocytes (11). Van der Vieren et al., using
d
2-expressing chinese hamster ovary (CHO) transfectants, demonstrated binding of
d
2 to a human intercellular adhesion molecule 3 (ICAM-3) chimeric protein (11).
Whether
d
2 is expressed on eosinophils, and how it
functions on these cells, were not examined.
The goal of these studies was to examine the expression
and function of d
2 integrins on human eosinophils. We
report that eosinophils express
d
2, that its surface expression can be acutely and chronically regulated by various
stimuli, and that, like
4 integrins, they can function as a
ligand for VCAM-1.
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Materials and Methods |
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Reagents.
The following murine IgG1 mAbs were used: irrelevant control IgG1 mAb (Coulter Corp., Hialeah, FL), CD11a mAb (MHM24; courtesy of Dr. James Hildreth, Johns Hopkins University School of Medicine, Baltimore, MD [12]), CD11b mAb (H4C2, Dr. Hildreth [13]; and clone 44, R&D Systems, Minneapolis, MN), CD11c mAb (BU-15; Immunotech, Inc., Westbrook, ME),Cell Isolation.
Normodense (specific gravityCHO Transfectants.
CHO cells were transfected with both the humanFlow Cytometry.
Expression of integrins on the CHO cell transfectants or on freshly isolated cells from blood after stimulation or culture was evaluated using single color indirect immunofluorescence and flow cytometry as described previously (17, 18). Dual color detection of basophils (using anti-IgE) and lower purity eosinophils in BAL fluids (using anti-CD9) was also performed. All samples were fixed in 1% paraformaldehyde (Sigma Chemical Co.) and analyzed using a flow cytometer (EPICS Profile II; Coulter Corp.). Approximately 10,000 events were collected and displayed on a 4-log scale, yielding values for mean fluorescence intensity (MFI).Adhesion Assays.
For eosinophils, both freshly purified and cultured, 51Cr-labeled cell adhesion to VCAM-1 (250 ng/ml) or BSA (1%)-coated wells was performed for 30 min at 37°C as described previously (20). In some experiments, cells were preincubated for 30 min at 4°C with saturating concentrations of one or more of the following blocking mAbs before examining their adhesion: CD18 (7E4), CD11a (MHM24), CD11b (clone 44), CD11c (BU-15),Statistical Analyses.
Statistical analyses were performed using an analysis of variance (ANOVA) with a Fisher post hoc t test. Significance was set at P < 0.05 for all tests. ![]() |
Results |
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Using
indirect immunofluorescence and flow cytometry, eosinophils were found to consistently express all four of the 2
integrins, including
d
2, with the rank order of MFI values
as follows: IgG control (3) < CD11c (15) <
d (22) <
4
(42) < CD11a (88) < CD11b (111) (values in parentheses
representative of n = 7). Eosinophils and neutrophils had
roughly similar levels of
d expression, whereas basophils
had approximately twice the levels of surface expression (n = 4, data not shown).
Subsequent studies were performed to determine whether
eosinophils could rapidly mobilize intracellular stores of
d
2 as has been reported for neutrophils (11). Purified
peripheral blood eosinophils were incubated for 15 min
with either PMA or the calcium ionophore A23187, and
the surface expression of several
chains of the
2 integrins was then measured by indirect immunofluorescence. Fig. 1 shows the kinetics of this upregulation with phorbol
ester. Both PMA (50 ng/ml) and calcium ionophore (1 µM, data not shown) significantly increased the expression
of
d integrin and CD11b. Within minutes of adding
PMA, expression increases, reaching significantly increased
levels by 10 min. Therefore, eosinophils appear to have preformed stores of
d
2 which, similar to CD11b stores,
can be rapidly mobilized to the cell surface. Other eosinophil-active stimuli were tested for their acute effects on
d
2 expression. Incubation of eosinophils for 15 min
with MDC (100 nM), IL-5 (10 ng/ml), RANTES (100 ng/ml), and Eotaxin (100 nM) failed to alter
d integrin
expression (data not shown).
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Many eosinophil responses can be enhanced by prolonged exposure to certain cytokines, such as IL-5, a phenomenon referred to as "priming" (22). Therefore, we determined whether eosinophil culture with IL-5 would lead
to changes in surface expression of d integrin. The kinetics of this effect on
d integrin expression is shown in Fig.
2. As can be seen, the level of
d integrin increases gradually, with statistically significant increases in levels at days
4-7 of culture. In contrast, levels of
4 integrin did not
change significantly. Because late phase BAL eosinophils
express many characteristics of cytokine-primed eosinophils (19, 23), their levels were also compared. Indeed, late
phase BAL eosinophils also showed a statistically significant
increase in the level of
d integrin expression, with levels
similar to those seen after 3 d of culture in IL-5 (Fig. 2,
right).
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Although d
integrin has been shown to bind ICAM-3 and mediate leukocyte-leukocyte adhesion (11), the next series of experiments were designed to examine other possible
d ligands
for eosinophils. In part because of previous studies suggesting
2 integrin-dependent, CD11b-independent eosinophil adhesion to VCAM-1 (20; and our unpublished observations), initial studies were performed using immobilized recombinant VCAM-1.
As shown in Fig. 3 A, freshly isolated eosinophils adhered to VCAM-1, and mAb blockade of 4 integrin effectively inhibited adhesion, whereas CD11b blockade had
no effect. However, adhesion could also be significantly
and consistently inhibited by the
d mAb 240I, albeit to a
lesser degree (~30% inhibition). Even more striking were
results of VCAM-1 adhesion experiments in which IL-5-
cultured eosinophils were used. Data in Fig. 3 B show that under these conditions, mAbs to CD18,
d, or
4 integrins were equally effective in reducing adhesion to background levels, whereas a combination of blocking mAbs to
CD11a, CD11b, and CD11c had no effect. Note also that
IL-5-cultured eosinophils displayed enhanced background
adhesion and reduced VCAM-1 adhesion compared with
those seen with freshly isolated eosinophils.
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To further verify that d
2 functions as a ligand for
VCAM-1, we generated CHO transfectants expressing the
human
d and
2 integrin chains and used them in adhesion assays. These transfected cells, unlike the control CHO
cells, expressed both the
d and
2 integrin chains at levels
similar to that on eosinophils (depending on passage number: an MFI for each subunit of 10-50, compared with an
MFI using an irrelevant IgG control mAb of 0.4-1.0; n = 5). Transfected CHO cells expressing
d
2 adhered to
VCAM-1-coated wells (16.0 ± 3.6% adhesion, mean ± SEM; n = 11), and adhesion was effectively blocked by an
F(ab')2 mAb against the first domain of VCAM-1 (80.1 ± 5.2% inhibition; P < 0.0005 vs.
d
2-transfected CHO
cell adhesion to VCAM-1, n = 3) as well as by mAbs (n = 3) against either CD18 (79.8 ± 18.1% inhibition; P < 0.0005) or
d integrin (59.6 ± 21% inhibition; P < 0.001). In contrast, control nontransfected CHO cells failed
to adhere to VCAM-1 (1.9 ± 0.9% adhesion; n = 3), and
neither the transfected nor the control CHO cells displayed
significant adherence to wells coated with another adhesion
protein, E-selectin (6.1 ± 3 and 1.5 ± 0.5% adhesion respectively, n = 2-5).
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Discussion |
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These studies have shown that d
2, like other
2 integrins, is expressed on human eosinophils, basophils, and
neutrophils. On peripheral blood eosinophils, the level of
d integrin expression is similar to that of
4 integrins,
greater than that of CD11c, and less than that of CD11a
and CD11b. Stimuli such as PMA and the calcium ionophore A23187 rapidly upregulated eosinophil surface expression of
d integrins, whereas a more gradual increase in surface expression was seen after 4-7 d of culture in media
containing IL-5. In adhesion assays,
d
2 integrin was
shown to function as a ligand for VCAM-1 in both freshly
isolated and IL-5-cultured eosinophils; these results were
corroborated in adhesion assays using
d
2-transfected CHO
cells. Based on mAb blocking studies with freshly isolated
eosinophils, adhesion to VCAM-1 was mainly mediated
through
4 integrins, the other known ligand for VCAM-1. However, in IL-5-cultured eosinophils, adhesion to
VCAM-1 was equally mediated by
4 and
d integrins.
Together, these data are the first to demonstrate activation-dependent regulation of
d
2 integrin expression and
function on human eosinophils and document a novel
function for eosinophil
d
2 as an alternative ligand for
VCAM-1.
There appear to be preformed stores of d integrin in
eosinophils, as evidenced by the rapid upregulation of
surface expression with exposure to PMA or calcium ionophore. These results are similar to those observed for
d
integrin and neutrophils (11). The kinetics of enhanced expression with PMA exposure was similar to that of CD11b,
suggesting that these two leukointegrins might exist in similar or identical intracellular compartments. The location of
this compartment for either integrin in eosinophils is not
known; however, in neutrophils, preformed stores of
CD11b have been localized to specific granules (24, 25).
The immunolocalization of these preformed
2 integrin
pools, as well as effects of more physiologic activators of
eosinophils on integrin expression, are currently under investigation.
In contrast to the rapid mobilization by PMA, a gradual
increase in surface d integrin expression was seen during
IL-5 culture. Whether this represents events occurring at
the level of transcription or translation, rather than slow
mobilization from preformed pools, is not yet known, due
in part to difficulties encountered in isolating eosinophil
mRNA as well as adverse effects of inhibitors of transcription and translation on eosinophil survival. In examining
levels of
d integrin on late phase BAL eosinophils, which
have already undergone cell adhesion and migration to get
to the airway lumen, levels of expression intermediate to
those seen on freshly isolated and IL-5-cultured eosinophils
were observed. These data suggest that at least a portion of
the elevated levels of
d found after IL-5 culture are likely
due to increased transcription and translation of
d integrin.
A particularly novel aspect of this study was the determination that d
2 integrin, expressed on eosinophils and
CHO transfectants, can function as a ligand for VCAM-1.
Although the exact binding site on VCAM-1 is unknown,
it is interesting that an mAb to the
4 integrin binding site
in the first domain of VCAM-1 completely blocked
d
2
integrin-dependent VCAM-1 adhesion. While this suggests that the
d
2 binding site is near or identical to that
for
4 integrins, additional studies are required to examine
this issue more directly. Whether
d
2 integrins can bind
to other
4 integrin ligands, such as fibronectin or mucosal
addressin cell adhesion molecule 1, is unknown. The finding that
d
2 integrin can function as a ligand for VCAM-1
appears to conflict with data presented in the first report
on human
d
2 integrin by Van der Vieren et al. (11). In
that paper it was shown that these same
d
2-transfected CHO cells bound to a soluble ICAM-3 construct but not
to a VCAM-1-Ig chimeric protein. Possible explanations
for this discrepancy include a lower affinity for soluble
ligand binding as well as other differences in assays, such as
temperature.
Depending on the experimental conditions, both 4 and
d integrins can mediate eosinophil adhesion to VCAM-1.
As we have shown in IL-5-cultured eosinophils, the relative contribution of
d integrin-dependent versus
4 integrin-dependent adhesion increases in parallel with an increase in
d integrin surface expression. However, because
regulation of integrin affinity also influences adhesive function, additional studies are needed to define the mechanisms responsible for these IL-5-induced changes. Another potential paradox from our findings is that although neutrophils express
d
2, they do not adhere to seven-domain
VCAM-1. The most plausible explanation for this again
appears to be related to integrin activation state. Freshly
isolated eosinophils and neutrophils express similar levels of
d integrins and, at least for eosinophils, much lower
d integrin-dependent adhesion responses than those seen with
activated cells. Whether this can be overcome with neutrophil activation is not yet known. Finally, the function of
d
integrin in vivo is currently under investigation. Based on
our results, it is plausible that because
d integrins on eosinophils bind to VCAM-1 and are upregulated with IL-5 in
vitro and in BAL fluids in vivo, this leukointegrin may play
a role in cytokine-primed eosinophil recruitment to inflammatory sites. However, evaluation of this hypothesis
will require further investigation.
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Footnotes |
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Address correspondence to Bruce S. Bochner, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. Phone: 410-550-2131; Fax: 410-550-2130; E-mail: bbochner{at}welchlink.welch.jhu.edu
Received for publication 25 June 1998 and in revised form 8 September 1998.
This work was supported in part by grants HL49545 and AI41472 from the National Institutes of Health and by a Developing Investigator Award to B.S. Bochner from the Burroughs Wellcome Fund.
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