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Blood, Vol. 93 No. 4 (February 15), 1999:
pp. 1406-1412
Previous Uptake of Apoptotic Neutrophils or Ligation of Integrin
Receptors Downmodulates the Ability of Macrophages to Ingest Apoptotic
Neutrophils
By
Lars-Peter Erwig,
Sharon Gordon,
Garry M. Walsh, and
Andrew J. Rees
From the Department of Medicine and Therapeutics, University of
Aberdeen, Aberdeen, UK.
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ABSTRACT |
Clearance of apoptotic neutrophils (polymorphonuclear leukocyte
[PMN]) by macrophages is thought to play a crucial role in resolution
of acute inflammation. There is increasing evidence that ingestion of
apoptotic cells modulates macrophage behavior. We therefore performed
experiments to determine whether ingestion of apoptotic PMN modulated
the uptake process itself. Rat bone marrow-derived macrophages (BMDM)
ingested apoptotic PMN by a process that was enhanced by tumor necrosis
factor (TNF) and attenuated by interferon (IFN)- , interleukin
(IL)-4, and IL-10. It was inhibitable by the tetrapeptide
arg-gly-gln-ser (RGDS), therefore implicating the
 v 3/CD36/thrombospondin pathway. Interaction of apoptotic PMN with
BMDM for 30 minutes, 48 hours before rechallenge reduced uptake of
apoptotic PMN by 50% compared with previously unchallenged BMDM.
Blocking initial uptake with RGDS abrogated the effect of preexposure.
Comparable and sustained attenuation of uptake was obtained by ligating
v3 with the monoclonal antibody (MoAb), F11, after a delay of
more than 90 minutes, whereas MoAbs to CD25 and CD45 had no effect.
Ligation of 61 and 12, integrins not previously implicated
in the engulfment of apoptotic cells also decreased uptake with similar
kinetics to F11. Therefore, apoptotic PMN regulate their own uptake
through an integrin-dependent process, which can be reproduced by
ligation of other integrins expressed by macrophages.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
MACROPHAGES INFLUENCE almost all aspects
of immunological and inflammatory responses and play an essential role
in linking the innate and acquired immunity.1 Macrophages
not only induce injury, but also control key events in the resolution of inflammation and the repair processes that follow it. One of the
critical functions in this process is phagocytosis of apoptotic cells
via specific recognition mechanisms. To date, a number of recognition
mechanisms for apoptotic cells have been described: (1) an
uncharacterized lectin-dependent interaction2; (2) a complicated charge sensitive process involving the CD36/vitronectin receptor ( v3) complex on the macrophage surface interacting with
unknown moieties on the apoptotic polymorphonuclear leukocyte (PMN)
surface via a thrombospondin bridge3,4; (3) a
stereo-specific recognition of phosphatidylserine that is expressed on
the surface of the apoptotic cell after loss of membrane
asymmetry5,6; (4) macrophage scavenger
receptors7; (5) the lipopolysaccharide (LPS)
receptor CD148-10 and macrosialin or CD68.11,12
The specific removal of apoptotic thymocytes,13
eosinophils,14 and neutrophils15 by macrophages
has been well described. Extensive tissue damage and inflammation both
precede and follow neutrophil death by necrosis. The cellular debris is
phagocytosed by macrophages, which are activated by the process. In
contrast, apoptosis of neutrophils is associated with the swift
recognition of intact cells by macrophages followed by their ingestion
and degradation. Local inflammation and tissue injury are avoided not
only because neutrophils are prevented from releasing their toxic
contents, but also because the macrophages usual proinflammatory secretory response to phagocytosis is not activated16 and
may be biased towards release of the anti-inflammatory cytokine
transforming growth factor (TGF)-.17
These results suggest that uptake of apoptotic neutrophils by
macrophages does not merely fail to induce synthesis of proinflammatory cytokines, but actively modulates macrophage function and biases the
profile of cytokines they release. This raises the question whether
uptake of apoptotic cells "imprints" a pattern of behavior on
macrophages analogous to the effect of exposure to some
cytokines.18 The specific purpose of the experiments
described here was to ascertain whether uptake of apoptotic neutrophils
modulates the ability of macrophages to ingest a second challenge with
apoptotic PMNs. The results show a substantial reduction in the
proportion of macrophages that ingest a second challenge of apoptotic
PMNs, but that uptake can still be modulated appropriately by
cytokines. The bone marrow-derived macrophages (BMDM) uptake of
apoptotic PMN is RGDS-dependent and presumptively occurs by the
v3/CD36/thrombospondin pathway. After a delay of at least 90 minutes, ligation of v3 also downmodulates uptake of apoptotic
cells specifically, and ligation of two other integrins, 61 and
12, have the same effect. This shows that uptake of apoptotic
cells is regulated by events that induce signalling through integrin
receptors irrespective of whether or not they are directly associated
with uptake. This raises the question whether uptake of apoptotic cells
via v3 reciprocally influences functions of integrins responsible
for cell adhesion and facilitate the emigration of macrophages from an
inflamed focus as described by Bellingan et
al.19
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MATERIALS AND METHODS |
Reagents.
Recombinant human tumor necrosis factor (rhTNF)-, rhTGF-, and
recombinant rat interferon (IFN)- were obtained from Boehringer (Ingelheim, Germany), Sigma Chemical Co (Dorset, UK), and Bradsure Biologicals Ltd (Loughborough, UK), respectively. Recombinant rat
interleukin (IL)-4 was produced in-house as described
previously20 using a Chinese hamster ovary
(CHO) cell line generously donated by Dr Neil Barclay (MRC
Cellular Immunology Unit, Oxford, UK). The rat monoclonal
antibody (MoAb), F11, against the integrin 3 chain21 was
a gift from Prof Michael Horton (Bone and Mineral Centre, University
College London Medical School, London, UK). The mouse
antirat integrin antibody 61, CD18, CD116, anti-CD45, anti-CD25,
anti-ED3, and mouse antihuman CD21 were obtained from Serotec (Oxford,
UK). The rabbit antihuman erythrocyte membrane antibody was obtained
from DAKO (Glostrup, Denmark). The tetrapeptides arg-gly-asp-ser
(RGDS), arg-gly-glu-ser (RGES), and phospho-L-serine were obtained from
Sigma Chemical Co.
Isolation and culture of BMDM.
Rat BMDM were obtained using a technique previously described in
detail.22 Briefly, bone marrow cells were flushed
aseptically from the dissected femurs of male Spraque Dawley rats with
a jet of complete medium directed through a 25-gauge needle to form a
single cell suspension. The cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) containing 2 mmol/L glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin, 10% heat-inactivated fetal
calf serum, and 10% L929 conditioned medium as a source of
macrophage-colony stimulating factor (M-CSF). After 7 days in culture,
BMDM were dispensed into 24-well culture plates (Corning, Corning, NY)
at a concentration of 5 × 105 cells/well and rested
in medium without added M-CSF for 24 hours before use in experiments.
Inhibition of uptake of apoptotic neutrophils.
BMDM were incubated with a series of inhibitors at concentrations of 1 mmol/L for 15 minutes at 4°C and were washed immediately before
interaction with apoptotic neutrophils. Phospho-L-serine was used as a
stereo-specific inhibitor of the macrophage phosphatidylserine receptor, using conditions described by Fadok et al.5 The
tetrapeptide arg-gly-asp-ser (RGDS) was used as described,4
and the noninhibitory peptide arg-gly-glu-ser (RGES) added as a control.
Assay for uptake of apoptotic neutrophils.
BMDM were transferred to 24-well plates at a density of 5 × 105 cells/well and rested for 24 hours before the medium
was changed and the cells incubated with various cytokines. Uptake of
apoptotic neutrophils was assessed after 48 hours using a
microscopically quantified phagocytic assay, which has previously been
described and illustrated in detail.4,23 Apoptotic
neutrophils were prepared from PMN isolated from fresh heparinized
normal human blood by dextran sedimentation and Percoll centrifugation.
They were aged in teflon bags for approximately 24 hours in RPMI 1640 supplemented with antibiotics and 10% fetal calf serum. More than 98%
of these cells excluded trypan blue while apoptosis was verified by oil
immersion light microscopy of May-Giemsa-stained cytospin preparations
as previously described.23 The apoptotic cells were washed
once and resuspended in RPMI at a concentration of 2.5 × 106/mL. A total of 1 mL of cells was added to each well and
allowed to interact with the macrophages for 30 minutes at 37°C in
a 5% CO2 atmosphere. The wells were washed in saline at
4°C to remove noningested PMN, fixed with 2% gluteraldehyde in
0.9% saline, and stained for myeloperoxidase to identify ingested PMN.
The proportion of macrophages that had ingested neutrophils was then counted by inverted light microscopy.
To determine the effect of previous ingestion of apoptotic neutrophils
on macrophages, rat BMDM were transferred to 24-well plates at a
density of 5 × 105 cells/well and rested for 24 hours
before the medium was changed and cells were incubated with either
medium alone, RGDS peptide followed by apoptotic neutrophils, or
apoptotic neutrophils alone. After 30 minutes incubation, the wells
were washed in saline at 4°C to remove noningested PMN, and the
macrophages were rested for 48 hours in control medium or medium
containing various cytokines. They were then reincubated for 30 minutes
with a second challenge of apoptotic neutrophils and the proportion of
macrophages that took up PMN assessed. The assay for uptake of
opsonized erythrocytes was performed exactly as previously
described.24
Ligation of the integrin receptors.
MoAbs to v3, 61, 12, CD25, and CD45 were used to
assess the effect of ligation of macrophage cell surface receptors on
uptake of apoptotic neutrophils. Macrophages were incubated at various
MoAb concentrations ranging from 0.01 to 10 µg/mL, for
30 minutes, at 4°C in saline, or were incubated with various concentrations of mouse antihuman CD21 as an irrelevant isotype-matched control. The cells were then washed before incubation in medium for
various times before the start of the standard interaction assay with
apoptotic PMN.
Quantitation of nitric oxide (NO) generation.
Generation of NO was measured by assaying culture supernatants for
nitrite, a stable reaction product of NO. Aliquots of 200 µL of each
cell-free culture supernatant were incubated with 50 µL of Griess
reagent (0.5% sulphanilamide, 0.05% N-(1-naphtyl) ethylendiamine
dihydrochloride in 2.5% phosphoric acid) in 96-flat-bottomed tissue
culture plates for 10 minutes at room temperature. The optical
densities of the assay samples were then measured at 540 nm using a
solution of phenol red free DMEM. In most experiments, nitrite was
measured 48 hours after exposure to cytokines.
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RESULTS |
Cytokines regulate uptake of apoptotic neutrophils by BMDM.
The initial experiment was designed to confirm our previous
observations that pro and antiinflammatory cytokines influence uptake
of apoptotic human neutrophils by uncommitted rat BMDM.20 TNF caused a 36% increase in the proportion of BMDM that took up
apoptotic neutrophils compared with controls, whereas IL-4, IL-10, and
IFN- inhibited uptake by 56%, 22%, and 42%, respectively, and
TGF- had no effect (Table 1). Incubation
with cytokines modulated not only the number of macrophages taking up
apoptotic cells, but also the average number of neutrophils per
macrophage, ie, IL-4 caused a 56% decrease in the number of
macrophages taking up apoptotic neutrophils and a 40% reduction in the
number of neutrophils per macrophage. These findings differ from those
reported for human monocyte-derived macrophages. In these cells,
incubation with proinflammatory cytokines (IFN and TNF) increased their
ability to ingest neutrophils, whereas antiinflammatory cytokines
(IL-4, IL-6, and IL-10) had no effect.25 These differences
could reflect the source and species of the macrophages used or the
conditions in which they were matured. Recently, Bonder et
al26 have shown that human 7-day-cultured monocytes did
not express the functionally active IL-2 receptor -chain, a
component of the IL-4 receptor, whereas macrophages did, which may
explain the different effect of IL-4 on uptake of apoptotic neutrophils
by monocyte-derived macrophages and BMDM.
BMDM use an integrin-dependent mechanism to recognize apoptotic PMN.
Human monocyte-like cell lines and murine peritoneal macrophages use
the phosphatidylserine receptor (PSR) for recognition of apoptotic
cells.27 Human monocyte-derived macrophages and murine BMDM
have been reported to use the v3/CD36/thrombospondin pathway.3 In our studies, 1 mmol/L RDGS specifically
inhibited uptake of apoptotic PMN by unstimulated rat BMDM and by
macrophages incubated for 48 hours with IFN-, TNF, IL-4, or TGF-.
Neither the control peptide RGES, nor phospho-L-serine, which inhibits PS-mediated recognition of apoptotic cells,5 had any effect on uptake by cytokine-stimulated or unstimulated macrophages
(Table 2). Thus, both uncommitted or
cytokine-stimulated rat BMDM use an integrin-dependent recognition
mechanism, presumptively the CD36/v3/thrombospondin system rather
than a PSR-dependent mechanism. This conclusion is strengthened by the
demonstration of v3 on the surface of BMDM by immunofluorescence
using the MoAb, F11, directed against the 3 subunit of the receptor
(data not shown). To verify the recognition mechanism, it would be
necessary to block either CD36 or v3 on the macrophage surface.
To our knowledge, the one antirat antibody available for this purpose
is the mouse MoAb F11 against the 3 subunit of the vitronectin
receptor, which is a poor blocking antibody under our experimental
conditions.
BMDM incubated with F11 for 45 minutes and then seeded in
vitronectin-coated plates adhered as efficiently as control
macrophages. There was no difference in the number of nonadherent cells
(less than 1% of the seeded cells in both groups) when aliquots of the supernatants of control and F11-treated macrophages were examined 2, 4, 12, and 24 hours after seeding (data not shown).
Previous uptake of apoptotic PMNs reduces the ability of BMDM to
ingest apoptotic PMN.
To determine the effect of uptake of apoptotic neutrophils on
macrophage function, uncommitted rat BMDM were challenged for 30 minutes with apoptotic neutrophils in medium alone or in the presence
of RGD peptide to prevent uptake. They were then rested for 48 hours in
medium before being reexposed to freshly prepared apoptotic
neutrophils. Macrophages that had previously ingested apoptotic PMN had
a markedly reduced ability to engulf apoptotic neutrophils compared
with control macrophages (Fig 1),
whereas their ability to take up opsonized erythrocytes was unchanged (data not shown). The difference cannot be attributed to a nonspecific effect of the neutrophils because macrophages challenged with PMN in
the presence of RGDS-peptide retain their subsequent ability to take up
apoptotic neutrophils (Fig 1).
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| Fig 1.
Figure 1 shows the percentage uptake of apoptotic
neutrophils by BMDM. The macrophages were incubated 48 hours before the
interaction assay with apoptotic neutrophils, RGDS followed by
apoptotic neutrophils or medium. They were then washed and cultured in
medium containing cytokines or medium alone before washing and a
30-minute interaction with apoptotic PMN; mean ± standard error (SE),
n = 10; * P < .01.
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The degree of inhibition was comparable to that observed when BMDM are
exposed to IFN-, IL-4, or IL-10, which we have previously shown
cannot be reversed by treatment with TNF.28 By contrast, uptake of apoptotic cells by BMDM did not abrogate the modulatory effects of TNF or other cytokines on uptake of apoptotic cells when
added to the medium after the initial challenge. However, in each,
their capacity to take up apoptotic PMNs was reduced by 50%.
Furthermore, prior uptake of PMNs did not affect the ability of IFN-
to prime macrophages for generation of NO
(Table 3). In this set of experiments,
there was no significant difference in IFN/TNF-induced NO generation
between uncommitted BMDM, macrophages that had ingested apoptotic
neutrophils, and macrophages that have been incubated with RGDS peptide
followed by apoptotic neutrophils. Thus, uptake of apoptotic cells
specifically inhibits BMDM ability to engulf apoptotic cells without
interfering with their ability to respond to a range of pro and
antiinflammatory cytokines.
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Table 3.
Effect of Uptake of Apoptotic PMN or Ligation of
Integrins on IFN/TNF-Induced NO Generation (Arbitrary Units)
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Ligation of the v3 receptor and other
integrins reduce uptake of apoptotic PMNs.
Overloading the macrophage phagocytic capacity provides the most
obvious explanation as to why uptake of apoptotic cells prevented further uptake. However, this seems unlikely for three reasons. First,
uptake of opsonized erythrocytes did not downmodulate the ability of
macrophages to ingest apoptotic neutrophils 48 hours later
(Table 4). Second, incubation of BMDM with
neutrophils from different donors known to induce high or low uptake
showed that irrespective of whether 20% or 40% of macrophages took up apoptotic cells and regardless of substantial differences in the number
of PMN ingested per macrophage, the degree of downmodulation was about
50% (data not shown). Finally, the 48 hours between the PMN challenges
should be sufficient to allow the macrophages to recover. The fact that
inhibition was prevented by incubation in the presence of RGDS
suggested that the mechanism might involve the
v3/CD36/thrombospondin pathway.4 This was addressed
by incubation of BMDM for 30 minutes with various concentrations of the
MoAb, F11, against the 3 subunit of the v3
receptor.21 F11 blocks the calcium response after peptide
binding to the vitronectin receptor in rat osteoclast.29 It
binds to v3 on the surface of macrophages, but did not alter
their adhesion to vitronectin, nor did it block uptake of apoptotic PMN
by BMDM. Despite this, ligation of v3 with F11 12 to 36 hours
before the interaction assay caused substantial reduction of PMN uptake
(Fig 2), comparable to that induced by
apoptotic PMN themselves.
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Table 4.
Effect of Uptake of Opsonized Erythrocytes on the Number
of Macrophages That Take up Apoptotic Neutrophils 48 Hours Later
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| Fig 2.
Figure 2 shows the effect of ligation of v3 (1 µg/mL) and an isotype-matched control CD21 (10 µg/mL) on uptake of
apoptotic neutrophils by BMDM. The macrophages were incubated for 30 minutes at various times before the start of the interaction assay.
Mean (percentage of uptake) ± SE, n = 8. * P < .01.
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These data indicate that ligation of v3 integrin decreases uptake
of apoptotic neutrophils after a delay of at least 90 minutes, whereas
an isotype-matched control mouse antihuman CD21 MoAb, which recognized
neither PMNs nor macrophages, had no effect. To examine the specificity
of the effects of F11, the experiments were repeated, first using MoAb
against 61, another integrin receptor expressed by macrophages,
but not known to be involved in recognition of apoptotic
neutrophils, and secondly using a MoAb against CD45, another
molecule on the macrophage plasma membrane. Strikingly the MoAb against
61 also downregulated uptake of PMNs with identical kinetics to
antibodies against v3, whereas the MoAb against CD45 had no
effect (Fig 3). Thus, ligation of integrins, but not of other receptors on the surface of
macrophages, specifically downregulates uptake of apoptotic cells after
a delay of more than 90 minutes, but did not affect the uptake of other particles such as opsonized red blood cells (data not shown).
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| Fig 3.
Figure 3 shows the effect of ligation of 61 and
CD45 (10 µg/mL) on uptake of apoptotic neutrophils by BMDM. The
macrophages were incubated for 30 minutes at various times before the
start of the interaction assay. Mean (percentage of uptake) ± SE,
n = 8. * P < .01.
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To confirm these observations, we performed another set of experiments
examining the role of receptor ligation on uptake of apoptotic PMNs by
macrophages after ligation of the receptors for 45 minutes 12 hours
before the interaction assay. Ligation of the integrin receptors CD11b
(percentage uptake 14.4 ± 2.1, P < .01) and CD18 (13.4 ± 3.4, P < .01) significantly decreased uptake of
apoptotic cells by macrophages, whereas incubation with MoAbs against
the IL-2 receptor CD25 (28.7 ± 3.5) present on the macrophage
surface, or ED3 (29 ± 4), only expressed by activated or tissue
macrophages, were not different from controls (29.8 ± 3.4). Thus,
ligation of three different integrin receptors specifically downmodulated macrophage ingestion of apoptotic neutrophils, whereas ligation of two other receptors on the macrophage surface did not. It
seems likely that at least some of the modulating effects of apoptotic
PMNs themselves can be attributed to this mechanism. In addition, there
was no significant difference in IFN/TNF-induced NO generation between
uncommitted BMDM and macrophages, which have been incubated with
antibodies that ligate their integrin receptors (Table 3). Thus,
similar to uptake of apoptotic cells, ligation of integrin receptors
specifically inhibits BMDM ability to engulf apoptotic cells without
interfering with their ability to respond to a range of pro and
antiinflammatory cytokines.
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DISCUSSION |
The specific uptake of apoptotic neutrophils by macrophages is one of
the critical steps in the resolution of inflammation.30 It
provides a way to remove neutrophils before granulocyte lysis and
release of the neutrophils' cytotoxic contents16 and does not activate the macrophages usual proinflammatory response to phagocytosis. Indeed, Fadok et al17 have recently provided
evidence that uptake of apoptotic cells induces macrophages to
synthesize the antiinflammatory cytokine, TGF-. The importance of
the process is illustrated by the observation that insufficient or
impaired capacity for phagocytic clearance leads to disintegration of
the cells undergoing apoptosis and worsening of tissue
damage.31 We hypothesized that alterations in the process
responsible for removal of apoptotic neutrophils might contribute to
these observations. In some situations, induction of a single episode
of acute inflammation resolves quickly, whereas a second episode
results in progressive tissue damage. One explanation for this might be
that the difference was caused by a reduced capacity to remove
apoptotic neutrophils.32 This prompted us to analyze the
effect of ingestion of apoptotic cells on the ability of macrophages to
take up a second pulse of apoptotic cells 48 hours later. The results
show that uptake of the second pulse 48 hours after the first is
consistently reduced by 50%, which could have a substantial effect on
tissue repair.
The characteristics of the mechanisms responsible for impaired uptake
demonstrate that it involves specific interaction between the
neutrophils and macrophages: (1) neutrophil uptake by BMDM was
inhibited by RGDS, which interrupts integrin-dependent recognition; (2)
it was not influenced by uptake of opsonized erythrocytes and was
independent of the magnitude of the first "neutrophil meal" and
thus unlikely to be due simply to macrophage "indigestion"; (3)
the effect was sustained for at least 48 hours; (4) it did not
interfere with cytokine-induced modulation of uptake of apoptotic cells; and (5) uptake of apoptotic neutrophils does not influence IFN-/TNF-induced generation of NO by macrophages. Taken together, these characteristics suggest that modulation is caused by the specific
interactions between macrophage receptors and ligands on the PMN.
A number of different macrophage receptor-mediated pathways have been
described to be involved in uptake of apoptotic neutrophils: (1) an
uncharacterized lectin-dependent interaction2; (2) a complicated charge sensitive process involving the CD36/vitronectin receptor (v3) complex on the macrophage surface interacting with
unknown moieties on the apoptotic PMN surface via a thrombospondin bridge3,4; (3) a stereo-specific recognition of
phosphatidylserine that is expressed on the surface of the apoptotic
cell after loss of membrane asymmetry5,6; (4) macrophage
scavenger receptors7; (5) the LPS receptor,
CD148-10 and macrosialin or CD68.11,12 Inhibition by RGDS, but not PS, suggests that uptake by rat BMDM in our
experiments is mediated by the v3/CD36/thrombospondin recognition
pathway, which has been extensively characterized by Savill et
al.3,4 The importance of v3 was identified in
blocking experiments using MoAbs. Our experiments were conducted using
the MoAb, F11, an antibody to the 3 chain, which blocks some
v3-dependent functions, but not the ability of BMDM to bind to
vitronectin under our experimental conditions. Despite this, it caused
a sustained downmodulation of the macrophages' ability to take up
apoptotic cells after a delay of more than 90 minutes, comparable in
degree to that seen after ingestion of apoptotic cells. This effect was
not observed with isotype-matched control antibodies or with antibodies
to CD25 or CD45 on the macrophage surface. Strikingly, however,
antibodies to three other integrin receptors, 61, CD11b, and
CD18, not known to be associated with uptake of apoptotic neutrophils,
had the same effect. Thus, ligation of 1, 2, and 3 integrins
all cause sustained specific downmodulation of uptake of apoptotic
cells, but no effect on uptake of opsonized erythrocytes, and
presumptively 1 and 2 integrins downmodulate the function of
v3 in neutrophil uptake.
There are precedents for cross-inhibition between integrin receptors,
including interactions involving v3. Blystone et
al33 have previously shown that ligation of v3 blocks
high-affinity phagocytic function, but not adhesive function of the
fibronectin receptor 51, possibly by influencing serine/threonine
kinase activity of the cytoplasmic portion 1 chain. Similarly
41 ligation inhibits 51-dependent expression of
metalloproteinases.34 Diaz-Gonzalez et al35 and
Fenczik et al36 have analyzed the cross-talk between
different integrins in detail and introduced the term transdominant
inhibition to describe this phenomenon. They showed that
ligation of llb3 suppresses adhesive properties of
51 and 21.35,36 They demonstrated that the
phenomenon was dependent on the assumption of the high-affinity state
of llb3 and was attributable to conformational
changes in the cytoplasmatic portion of the 1 chain.37
It is not yet clear at what level changes in integrins might modulate
uptake of apoptotic cells, partly because of uncertainties about the
signalling pathways involved.
The intracellular signalling pathways that control uptake of apoptotic
cells have not been studied systematically. However, Rossi et al38 have recently reported that
activation of cyclic adenosine monophosphate (cAMP) signalling pathways
by inflammatory mediators downmodulates macrophage ingestion of
apoptotic cells and that alteration in cAMP concentrations might be
responsible for the observation that ligation of CD44 specifically
enhances phagocytosis of apoptotic neutrophils.39 Our
results suggest that uptake is also regulated by cross-talk between
integrins and emphasize the multiple levels of control for macrophage
removal of apoptotic cells. The ability of macrophages to ingest
apoptotic cells can be dynamically regulated, providing a potential
target for modulation of inflammation.
Furthermore, our observations also have the important implication that
ligation of v3 caused by uptake of apoptotic cells might
downmodulate integrin-dependent macrophage adhesion and facilitate
migration of macrophages from an inflamed site to the local lymph
nodes, as has been described by Bellingan et
al.19 We are currently conducting experiments to test this possibility.
| |
ACKNOWLEDGMENT |
The authors are indebted to A. Woodger for preparing this manuscript.
| |
FOOTNOTES |
Submitted May 8, 1998; accepted October 13, 1998.
Supported by Grant No. ER 254/1-1 from the Deutsche
Forschungsgemeimschaft (to L.-P.E.), Grant No. 044988/2/95/2 from the Wellcome Trust (to G.M.W.), and the National Kidney Research Fund.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Lars-Peter Erwig, MD,
University of Aberdeen, Department of Medicine and Therapeutics,
Institute of Medical Sciences, Foresterhill, Aberdeen AB25 2ZD, UK;
e-mail: L.P.Erwig{at}abdn.ac.uk.
| |
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Abstract
Introduction