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Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI 48109; and
Pulmonary and Critical Care Medicine Section, Medical Service, Department of Veterans Affairs Medical Center, Ann Arbor, MI 48105
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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) clear apoptotic
lymphocytes poorly. To test this hypothesis, we compared in vitro
phagocytosis of apoptotic thymocytes by resident AM and peritoneal
macrophages (PM) from normal C57BL/6 mice. AM were deficient
relative to PM both in percentage containing apoptotic thymocytes
(19.1 ± 1% vs 96 ± 2.6% positive) and in phagocytic index
(0.23 ± 0.02 vs 4.2 ± 0.67). This deficiency was not due to
kinetic differences, was seen with six other inbred mouse strains, and
was not observed using carboxylate-modified polystyrene microbeads.
Annexin V blockade indicated that both M types cleared apoptotic T
cells by a mechanism involving phosphatidylserine expression. By
contrast, neither mAb blockade of a variety of receptors (CD11b, CD29,
CD51, and CD61) known to be involved in clearance of apoptotic cells,
nor the tetrapeptide RGDS (arginine-glycine-aspartic acid-serine)
blocked ingestion by either type of macrophage. To confirm these
studies, apoptotic thymocytes were given intratracheally or i.p. to
normal mice, and then AM or PM were recovered 30–240 min later.
Ingestion of apoptotic thymocytes by AM in vivo was significantly
decreased at all times. Defective ingestion of apoptotic lymphocytes
may preserve AM capacity to produce proinflammatory cytokines in
host defense, but could contribute to development of autoimmunity by
failing to eliminate nucleosomes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)3
are believed to be the primary phagocytes responsible for ingestion of
apoptotic cells in most organs (3). This process is
generally highly efficient, so that even in tissues with known high
rates of lymphocyte apoptosis such as the thymus, it has generally been
difficult to demonstrate apoptotic lymphocytes in vivo
(4). For this reason, it was somewhat surprising that we
were previously able to detect large numbers of apoptotic lymphocytes
in the lungs of mice, both during the immune response to particulate Ag
and even in normal mice (5). As a potential explanation,
we hypothesized that alveolar M (AM), the chief phagocyte of the
gas-exchanging regions of the lungs, might clear apoptotic T cells
poorly relative to other types of M.
Although they all ultimately derived from common hematopoietic
precursors, M within various tissues differ considerably in
morphology, biochemistry, secretory products, surface phenotype, and
function. In particular, AM are a distinctive cell type that reside
in the unique environment of the pulmonary alveolus, where they are
exposed to high ambient oxygen concentrations, to pulmonary surfactant
that is rich in both lipids and unique opsonins, and to a large daily
burden of inhaled particulates (6, 7). Because these
particulates must be cleared without compromising gas exchange through
excessive inflammation, it is likely that AM are specialized
phagocytes. Indeed, AM differ from other M populations in
expression of novel receptors (8), in Ag-presenting
capacity (9), and in eicosanoid production (10, 11). The capacity of murine AM to clear apoptotic lymphocytes
has not been described previously, although resident AM from normal
rabbits have been shown to ingest apoptotic human neutrophils less
avidly than inflammatory lung M recovered from lungs of rabbits
undergoing immune complex injury (12).
Clearance of apoptotic cells by phagocytes is a complex and
incompletely understood process that involves both recognition and
ingestion steps (13). Multiple receptors appear to be
involved in each of these steps, with the specific receptors
used depending both on the apoptotic target and on the
activation state of the phagocyte. Recognition of exposed
phosphatidylserine (PS) on the surface of apoptotic cells in a
stereo-specific fashion is an important mechanism for many apoptotic
targets, especially lymphocytes (14, 15, 16). The
receptors responsible for PS recognition have not yet been definitively
identified (17), but candidates include CD14 (a
glycosylphosphatidylinositol-linked receptor for LPS)
(18), CD36 (a receptor for thrombospondin also known as
the class B scavenger receptor), and CD68 (a receptor for oxidized low
density lipoprotein). Multiple adhesion receptors have also been
implicated in phagocytosis of apoptotic cells in various systems.
Phagocytosis of apoptotic neutrophils by human monocyte-derived M
(HMDM) has been shown to involve the vitronectin receptor (VNR)
(
vß3 integrin,
CD51/CD61), which together with CD36 on the M binds unidentified
ligands on the neutrophil via thrombospondin (19). Thus,
differential expression of a variety of surface receptors could
underlie difference between M subtypes in clearance of apoptotic
cells.
The goal of this study was to determine the ability of murine resident
AM to ingest apoptotic lymphocytes. As a standard model of apoptotic
T cells (20, 21), we used thymocytes induced to become
apoptotic by exposure to dexamethasone. We found that in comparison
with resident peritoneal M (PM), resident murine AM displayed
a marked and specific deficiency in the ability to ingest apoptotic
thymocytes both in vitro and in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following anti-murine mAbs were purchased from
PharMingen (San Diego, CA): 2D7 (anti-CD11a); M1/70
(anti-CD11b); HL3 (anti-CD11c); rmC5-3 (anti-CD14); 2.4G2
(anti-CD16/CD32); C71/16 (anti-CD18); Ha2/5 (anti-CD29);
R1-2 (anti-CD49d); 5H10-27 (anti-CD49e); H9.2B8
(anti-CD51); 2C9.G2 (anti-CD61); and R3-34 (control rat IgG1
); R35-95 (control rat IgG2a ); A95-1 (control rat IgG2b )
G235-2356 (control hamster IgG1 
); A19-4 (control hamster IgG3 );
A19-3 (control hamster IgG1 ); G235-1 (control hamster IgM).
Paramagnetic microbeads coated with monoclonal anti-murine CD19 or
anti-CD90 were purchased from Miltenyi Biotec (Auburn,
CA).
Mice
Pathogen-free inbred female mice were used in all experiments. C57BL/6 (H-2b) and C3H/HeNCrIRB (H-2k) mice were purchased from Charles River Laboratory (Wilmington, MA); AKR/J (H-2k), C57BL/10J (H-2b), C3H/HeJ (H-2k), and DBA/1J (H-2q) mice were purchased from The Jackson Laboratory (Bar Harbor, ME); BALB/c (H-2d) mice were purchased from Taconic Laboratories (Germantown, NY). Mice were purchased at 7–8 wk of age and used at 8–14 wk of age. Mice were housed in the Animal Care Facility at the Ann Arbor Veterans Affairs Medical Center, which is fully accredited by the American Association for Accreditation of Laboratory Animal Care, where they were fed standard animal chow (rodent lab chow 5001; Purina, St. Louis, MO) and chlorinated tap water ad libitum. This study complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare Publication (National Institutes of Health) 80-23) and followed a protocol approved by the Animal Care Committee of the local Institutional Review Board.
Isolation and culture of M
Mice were euthanized by asphyxia in a high
CO2 environment. AM were collected by
bronchoalveolar lavage (BAL) using a total of 10 ml Dulbecco’s PBS
(Life Technologies, Grand Island, NY) containing 0.5 mM EDTA. BAL was
performed in 1-ml aliquots with gentle massage of the thorax, as
previously described (22). Greater than 95% of BAL cells
were AM. PM were collected by peritoneal lavage using the same
type of PBS, which was administered in 2-ml aliquots to a total volume
of 10 ml. PM among the lavage cells were first enriched by negative
selection using CD19- and CD90-conjugated paramagnetic beads, according
to the manufacturer’s instructions. M were plated at 2 x
105 cells/well in sterile eight-well Lab-Tek
slides (Nalge Nunc International, Naperville, IL) and, after 1-h
incubation at 37°C, nonadherent cells were removed by gentle washing.
M monolayers were cultured overnight in complete medium (RPMI 1640
containing 25 mM HEPES, 2 mM L-glutamine, 1 mM pyruvate,
100 U/ml penicillin/streptomycin (all obtained from Life Technologies),
10% heat-inactivated FBS (triple filtered at 100 nm 
25 EU/ml
endotoxin) 25 mg/dl hemoglobin) (HyClone Laboratories, Logan,
UT), and 55 µM 2-ME (Sigma, St. Louis, MO)) in a 5%
CO2 environment at 37°C before use in the
phagocytosis assay.
Isolation and apoptosis induction in thymocytes and cloned T cells
Thymuses were harvested from normal mice and minced to yield a single-cell solution. To induce apoptosis, thymocytes were suspended with RPMI 1640 containing 10% heat-inactivated FBS at the concentration of 1 x 106/ml and incubated with a final concentration of 10-6 M dexamethasone (Sigma) overnight. Thymocytes were 50.9% early apoptotic and 42.1% late apoptotic, as demonstrated by simultaneous annexin V and propidium iodide staining and flow cytometric analysis. CTLL-2 cells were induced to apoptosis by deprivation from IL-2 for 16 h. The resulting preparation was 44.9% early apoptotic and 23.1% late apoptotic.
Isolation and apoptosis induction in neutrophils
Neutrophils were harvested from the peritoneum of mice that had been treated 16 h and again 3 h previously by i.p. injection of 1 ml of a 9% solution (w/v) of casein (Sigma) in PBS containing 0.9 mM CaCl2 and 0.5 mM MgCl2 (23). Neutrophils were purified from this peritoneal lavage using NIM-2 (Cardinal Associates, Santa Fe, NM), according to the manufacturer’s directions. Purity was 96.6% by differential cell count of a Giemsa-stained cytospin slide. Neutrophil apoptosis was induced by UV irradiation (254 nM) for 15 min, followed by overnight incubation in complete medium at 37°C. The resulting preparation was 19.7% early apoptotic and 33.4% late apoptotic, as judged by staining with annexin V-FITC plus propidium iodide and flow cytometric analysis.
Apoptosis assay
Leukocyte apoptosis was measured by flow cytometric analysis of surface expression of PS, a sensitive and specific measure of early apoptosis (15, 24). For this purpose, 100-µl aliquots were stained with annexin V-FITC (Apoptosis Detection Kit; R&D Systems; Minneapolis, MN), according to the manufacturer’s protocol. Cells were analyzed without fixation by flow cytometry within 1 h of staining.
Phagocytosis assays
Phagocytosis of apoptotic thymocytes in vitro was assayed by
adding 2 x 106 apoptotic thymocytes
suspended in 400 µl of complete medium to each well of the Lab-Tek
slides containing adherent M monolayers. Heat-inactivated serum
(HyClone) was included at a final concentration of 10% during the
coincubation, as phagocytosis of apoptotic thymocytes by resident PM
has been shown to be dependent on serum (21). The slides
were incubated for 1.5 h at 37°C, washed with ice-cold PBS
containing 0.5 mM EDTA, and stained using hematoxylin and eosin Y (H&E)
(Richard-Allan, Kalamazoo, MI). Phagocytosis was evaluated by counting
200–300 macrophages per well at 1000 magnification under oil
immersion. Results were expressed as percentage of M containing at
least one ingested thymocyte (percent phagocytic), and as phagocytic
index, which was generated by multiplying the percentage of
phagocytosis by the mean number of phagocytosed cells per
M.
As a control for the ability of M to ingest particles, 8 x
106 FITC-labeled carboxylate-modified polystyrene
microbeads (1.7 µm mean diameter) (catalogue 17687; Polysciences,
Warrington, PA) were coincubated with adherent AM or PM (2
x 105 cells in a final volume of 400 µl in
complete medium) at 37°C in a 5% CO2
environment for 90 min. The slide was then washed three times with PBS
containing 0.5 mM EDTA. Phagocytosis was determined immediately by
fluorescence microscopy under oil immersion.
Assay of in vivo phagocytosis
To assay the ability of M to ingest apoptotic cells in vivo,
5 x 107 apoptotic thymocytes in 50 µl
normal saline were injected either intratracheally or i.p. using the
methods previously described for Ag administration (25).
BAL and peritoneal lavage were collected 2 h later. Slides of
lavage cells were prepared by cytocentrifugation (Shandon, Pittsburgh,
PA) and stained with H&E.
Inhibition of phagocytosis of apoptotic cells
For each blocking experiment, mAbs were used at final
concentrations that were saturating as demonstrated by flow cytometry.
Monolayer M were incubated with specified mAb for 30 min at 4°C.
The cells were gently washed twice with PBS and then cocultured with
apoptotic thymocytes for 90 min at 37°C in complete medium. For
annexin-blocking experiments, apoptotic thymocytes were incubated with
purified human annexin V (40 µg/105 cells) in
binding buffer (10 mM HEPES/NaOH, pH 7.4, 0.14 M NaCl, 2.5 mM
CaCl2) for 15 min at room temperature. The cells
were added without washing to the M monolayers and were cocultured
for 90 min at 37°C.
Immunostaining and flow cytometry
M freshly isolated by BAL or peritoneal lavage were used to
analyze expression of receptors potentially involved in clearance of
apoptotic cells. M were washed twice in staining buffer (Difco,
Detroit, MI), resuspended in 100 µl staining buffer, and incubated
for 30 min at 4°C in the dark with labeled Abs diluted in 100 µl
staining buffer. Final Ab concentrations were 1–2
µg/106 cells. FcR was blocked using mAb 2.4G2
(anti-CD16/32) for all primary mAbs except rmC5-3, as binding of
this anti-CD14 mAb has been reported to be inhibited by 2.4G2
(26). After incubation, cells were washed twice in
staining buffer, resuspended in 0.5 ml staining buffer, and analyzed
immediately.
Flow cytometry was performed as previously described in detail (27) using a FACScan cytometer (Becton Dickinson, Mountain View, CA) running CellQuest software on a PowerPC microcomputer (Apple, Cupertino, CA) for data collection and analysis. A minimum of 10,000 viable cells was analyzed to determine cell-surface receptor expression.
Purification of recombinant annexin V
To produce large quantities of annexin V for use in blocking experiments, recombinant human annexin was purified as described by Krahling (28). TG1 strain Escherichia coli containing a plasmid encoding human placental annexin V (clone pRK6; American Type Culture Collection, Manassas, VA) was cultured overnight at 37°C in 200 ml LB medium containing 50 µg/ml ampicillin. After overnight incubation, this mixture was diluted 5-fold into 1 L of fresh LB medium and was cultured for an additional 3 h. Next, isopropyl ß-D-thiogalactopyranoside was added to a final concentration of 1 mM. After 4 h of additional growth, bacteria were harvested by centrifugation (5,000 x g, 4°C, 15 min), and the pellet was resuspended in an equal volume of spheroplast buffer (0.5 mM EDTA, 750 mM sucrose, 200 mM Tris, pH 8). Lysozyme was added to a final concentration of 1 mg/ml immediately before the addition of 7-fold volume of spheroplast buffer diluted 1/1 with water and incubated for 30 min on ice. Spheroplasts were collected by centrifugation (14,000 x g, 4°C, 30 min), and the pellet was resuspended in 10 ml ultracentrifugation buffer (2 mM EDTA, 5 mM MgCl2, 100 mM NaCl, 0.1 mg/ml of RNase, 0.1 mg/ml of DNase I, 2 mM PMSF, 0.5 µg/ml of pepstatin A, 0.1% (w/v) Triton X-100, 20 mM Tris, pH 8). The suspension was centrifuged overnight at 100,000 x g at 4°C, and then the supernatant was harvested.
Liposomes for use in purification of the annexin V were prepared by dissolving 2 mg PS and 1 mg phosphatidylcholine (Sigma) in chloroform and drying the mixture under nitrogen gas. The lipid mixture, resuspended in 5 ml of liposome buffer (100 mM NaCl, 3 mM MgCl2, 20 mM Tris, pH 8) by vortexing, was sonicated for 10 min using a probe sonicator to prepare liposomes. These liposomes were added to the bacterial culture supernatant and calcium content was adjusted to 5 mM by addition of CaCl2. The mixture was incubated on ice for 30 min, and then was centrifuged at 40,000 x g for 45 min at 4°C. The pellet was washed once in washing buffer (5 mM CaCl2, 100 mM NaCl, 3 mM MgCl2, 20 mM Tris, pH 8) and resuspended in extraction buffer (10 mM EDTA, 100 mM NaCl, 3 mM MgCl2, 20 mM Tris, pH 8). The liposomes were removed by centrifugation for 1 h at 50,000 x g and 4°C. The supernatant was dialyzed in PBS, pH 7.4, and concentrated using a Centricon filter (Millipore, Bedford, MA). The purity of the protein was tested by SDS-PAGE and Coomassie staining, which indicated the product to be >90% pure.
Statistical analysis
Data were expressed as mean ± SEM. Statistical
calculations were performed using Statview and SuperANOVA programs
(Abacus Concepts, Berkeley, CA) on a Macintosh PowerPC G3 computer.
Continuous ratio scale data were evaluated by unpaired Student
t test (for two samples) or ANOVA (for multiple comparisons)
with post hoc analysis by the Tukey-Kramer test or by the two-tailed
Dunnett test, which compares treatment groups specifically to a control
group (29). Use of these parametric statistics was deemed
appropriate, as phagocytosis of apoptotic thymocytes by PM has been
shown to follow a Gaussian distribution (21). Percentage
data were arcsine transformed before analysis to convert them from a
binomial to a normal distribution using tables in Zar
(29). Significant differences were defined as
p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were deficient in phagocytosis of apoptotic thymocytes in
vitro relative to PM
Coculture of adherent AM and PM from normal C57BL/6 mice
with a 10-fold greater number of apoptotic thymocytes for various times
disclosed a marked deficiency in phagocytosis by AM (Fig. 1
). This deficiency was noted at all time
points, and was especially evident in the percentage of M that had
ingested even a single thymocyte. Considering results of several
experiments, 79–89% of PM were positive for phagocytosis in 60 min
vs only 3–12% of AM, and by 90–120 min a plateau in percentage of
positive M had essentially been reached by both cell types, with
over 90% of PM, but only 6–28% of AM, having ingested at least
one apoptotic cell. Phagocytic index also showed a large difference
between the two cell types, which continued to diverge through 6 h
of assay. Most PM ingested multiple apoptotic thymocytes, whereas
virtually no AM ingested more than a single thymocyte. Based on
these results, we selected 90 min for further analysis as a convenient
but sufficiently long duration of assay to detect differences between
the two M types.
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). Ingested cells
appeared intact, suggesting that some surface change promoted
phagocytosis of apoptotic thymocytes before their lysis. The observed
difference between the two M types could not be explained by
differences in viability or apoptosis of the M themselves (96.3
± 1.1% viable AM by trypan blue exclusion vs 97.1 ± 0.5%
viable PM, p = 0.51; 13.7 ± 1.9% apoptotic
AM by annexin-FITC binding vs 13 ± 3.4% apoptotic PM;
p = 0.87; mean ± SEM of four experiments). Nor
was the deficiency due to inhalation of a high
CO2 content during euthanasia (which we used
rather than the usual exsanguination while under deep anesthesia via
i.p. pentobarbital to permit harvest of AM and PM from the same
mice) (percentage of positive AM after CO2
asphyxia, 28.9 ± 3.2% vs 25.4 ± 1.7% positive after
pentobarbital euthanasia, p = 0.37; phagocytic index
after CO2 asphyxia, 0.35 ± 0.04 vs
0.29 ± 0.025, p = 0.25; n = 5
mice in each group). Additional control experiments demonstrated that
the relative deficiency of phagocytosis by AM did not result from
the overnight incubation step used (Table I). Furthermore, varying the ratio of
apoptotic thymocytes to macrophages to ratios as high as 100:1 did not
significantly increase phagocytosis by AM (data not shown). Hence,
artifacts of the in vitro assay did not appear to explain the
differences in phagocytosis between the two types of M.
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). In all cases, AM showed highly
significantly decreases in percentage of phagocytic M
(p < 0.001, all strains) and in phagocytic
index (p < 0.001, all strains) relative to
PM of the same strain. Interestingly, when results of AM were
compared between the mouse strains, both measures of phagocytosis by
AM of AKR and C57BL/10 mice were slightly but significantly higher
than those of AM of all other strains except DBA/1 (ANOVA with
Tukey-Kramer post hoc testing). No differences were seen between M
of C3H/HeN mice and those of C3H/HeJ mice, which has a naturally
occurring dominant-negative mutation in the innate immune receptor TLR4
(30). All remaining experiments were performed with M
of the C57BL/6 strain, which gave an intermediate result.
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were globally deficient in
phagocytic function, we examined the capacity of both types of M to
ingest two types of particles: opsonized zymosan and
carboxylate-modified polystyrene microbeads, a widely used model of
opsonin-independent phagocytic activities in vitro (31).
AM and PM had the same ability to ingest opsonized zymosan
(93.1 ± 2.4% phagocytic for AM vs 98.6 ± 0.3% for
PM, p = 0.09, unpaired t test; phagocytic
index 6.3 ± 0.5 for AM vs 7 ± 0.3 for PM,
p = 0.3, unpaired t test). The two types of
M also had identical phagocytosis of microbeads (62.4 ± 8.1%
phagocytic for AM vs 68.3 ± 4.6% for PM, p
= 0.56, unpaired t test; phagocytic index 3.1 ± 0.3
for AM vs 3.2 ± 0.2 for PM, p = 0.73,
unpaired t test). Thus, it appeared that the phagocytic
defect was specific for ingestion of apoptotic cells.
Because overnight incubation in dexamethasone allowed some of the
thymocytes to progress to late apoptosis, potentially limiting the
generalizability of our results, we performed a direct comparison with
ingestion of early apoptotic thymocytes induced by 6-h dexamethasone
incubation (42% annexin positive, 9% propidium iodide positive).
Markedly deficient ingestion of apoptotic thymocytes was again seen
using AM but not PM, with no difference between different
durations of dexamethasone incubation (for AM, 35% positive M
using 6-h treated thymocytes vs 33.8 ± 1.5% positive M using
18-h thymocytes, which were 57.1% propidium iodide positive,
p = 0.55, unpaired t test; phagocytic index
0.50 ± 0.03 for 6-h thymocytes vs 0.53 ± 0.03 for 18-h
thymocytes, p = 0.55, unpaired t test). In
the remainder of the experiments in the current study, we continued to
use overnight dexamethasone incubation. Experiments using two other
cell types as apoptotic targets indicated that the finding was not
limited to thymocytes. Resident murine AM also ingested apoptotic
murine neutrophils poorly in comparison with resident murine PM
(8.4 ± 0.9% phagocytic for AM vs 42.3 ± 11.8% for
PM, p < 0.02, unpaired t test;
phagocytic index 0.09 ± 0.01 for AM vs 0.52 ± 0.17 for
PM, p < 0.04, unpaired t test). The same
was true for the cloned T cell line CTLL-2, induced to apoptosis by
IL-2 deprivation (4.1 ± 1.7% phagocytic for AM vs 41.3
± 6% for PM, p < 0.001, unpaired t
test; phagocytic index 0.04 ± 0.02 for AM vs 0.49 ± 0.08
for PM, p < 0.001, unpaired t test).
Phagocytosis of apoptotic thymocytes was blocked by binding annexin V to apoptotic cells
A variety of mononuclear phagocytes, including
thioglycolate-elicited murine PM and human M cell lines, has been
shown to recognize apoptotic cells via expression of PS on the surface
of the apoptotic cell shortly after commitment to cell death. Although
the receptors responsible for this recognition event are currently
unknown, it is possible to block the process using annexin V itself
(28). Preliminary experiments showed that unconjugated
annexin V at 40 µg/105 thymocytes (final
concentration 200 µg/ml) would totally inhibit subsequent binding of
annexin-FITC, and that this concentration of annexin was not toxic to
macrophages during incubation for up to 16–18 h (data not shown).
Preincubation of apoptotic thymocytes with annexin V substantially
inhibited phagocytosis by adherent monolayers of both types of M
(Fig. 3). Phagocytosis (expressed as
percentage of positive M) was reduced by 84.7% in PM and by
83.9% in AM. The inhibitory effect of annexin was specific to
recognition of apoptotic cells; preincubation of carboxylate microbeads
with annexin V had no inhibitory effect on phagocytosis by either type
of M (for AM, 62.8% phagocytosis positive without annexin
preincubation vs 65.2%; phagocytic index 3.2 vs 3.1; for PM, 64.7%
phagocytosis positive without annexin preincubation vs 65.2; phagocytic
index 2.9 vs 3). Therefore, both types of murine M appeared to use
recognition of PS expression on apoptotic T cells as a critical signal
to initiate elimination.
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and PM differed in expression of surface receptors
potentially involved in clearance of apoptotic cells
To attempt to define the reason for the marked difference between
murine AM and PM in phagocytosis of apoptotic thymocytes, we next
examined surface expression of VNR (CD51/CD61) and of several other
integrins that might potentially be involved in clearance of apoptotic
cells (Fig. 4). Expression of
ß1 and ß2 integrins was
examined in this context due to recent reports of their role in
adherence to and phagocytosis of apoptotic leukocytes, respectively
(32, 33). The current analysis demonstrated several
differences in receptor expression between the two types of resident
M (Table III). Both AM and PM
had detectable surface expression of CD51, although expression was more
uniform and significantly greater on AM. However, expression of CD61
was much lower on AM than on PM, suggesting that AM must use
an alternative integrin ß-chain in conjunction with CD51. Both M
expressed CD11a, although levels were again more uniform and
significantly higher on AM. AM and PM had nearly reciprocal
expression of CD11b (which was high on PM and nearly absent from
AM) and of CD11c (which had the converse expression). Total
ß2 integrin expression by the two M types
was roughly equivalent, as judged by expression of the common ß-chain
CD18. Expression of CD29, the ß-chain common to the
ß1 integrins, was equivalent on the two types
of M. Relative to AM, PM had higher expression of CD49d, the
-chain of VLA-4, but not of CD49e, the -chain of VLA-6. Thus,
differences in one or more of these receptors were potential
explanations for the observed differences in phagocytosis of apoptotic
thymocytes.
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,
no single mAb significantly blocked either measurement of phagocytosis
of apoptotic thymocytes compared with treatment with isotype control
(Fig. 5A). Nor did several
combinations of blocking mAbs (CD29 plus CD49e; CD29 plus CD51; CD51
plus CD61) have a significant effect (Fig. 5A). For PM,
small but statistically significant effects were seen in some
experiments in percent of phagocytic M using anti-CD29 alone or
the combination of anti-CD29 plus anti-CD49e and anti-CD29
plus anti-CD51 and in phagocytic index using mAb against CD29 or
the combination of anti-CD29 plus anti-CD51 (Fig. 5B).
However, these changes were not seen in all experiments (data not
shown). To further test the importance of integrins in the ingestion of
apoptotic thymocytes by murine M, we preincubated adherent M with
either of two tetrapeptides, arginine-glycine-aspartic acid-serine
(RGDS) or arginine-glycine-glutamic acid-serine (RGES). RGDS is well
known to block a variety of integrins, whereas RGES serves as a
nonblocking control peptide (34). No inhibition of
phagocytosis was seen with either tetrapeptide (Fig. 6), providing further evidence against an
obligate role for VNR (or other arginine-glycine-aspartic acid-binding
integrins) in this system.
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types had low
expression of CD14 immediately after isolation (Table III
and Fig. 7A). Because it was
conceivable that this GPI-linked protein might have been released from
the M surface during isolation, we analyzed surface expression after
overnight incubation (i.e., at the time of assay for thymocyte
ingestion) on M released from incubation by washing with ice-cold
medium containing EDTA without enzyme treatment. This analysis showed
that CD14 expression could be induced on virtually all PM by
overnight incubation, whereas only a minority of AM expressed CD14
and that predominantly at levels only slightly above the staining with
isotype control mAb (Fig. 7A). No inhibitory effect on
thymocyte ingestion was seen using mAb rmC5-3 at a variety of
concentrations with either M type (Fig. 7, B and
C).
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and PM had different ability to engulf apoptotic cells
in vivo
Finally, to confirm the biologic significance of these in vitro
results, we examined clearance of apoptotic thymocytes in vivo.
Apoptotic thymocytes were administered intratracheally or i.p. to mice,
and then M were recovered by BAL or peritoneal lavage 30–240 min
later, and phagocytosis was determined. Results showed that AM
ingested very few apoptotic thymocytes at any time point, whereas
phagocytosis of apoptotic thymocytes by PM was readily detected
(Fig. 8). It was not feasible to
calculate the phagocytic index in these experiments, as PM appeared
to degrade ingested cells more rapidly than was noted in vitro. Thus,
AM were also markedly deficient in ingestion of apoptotic cells
relative to PM in vivo.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, resident murine AM are markedly deficient in ability
to ingest apoptotic thymocytes both in vitro and in vivo. This
deficiency appeared to be specific for apoptotic cells, as AM
ingested opsonized zymosan and carboxylate-modified polystyrene
microbeads as well as did PM. Annexin V blockade produced near total
inhibition of ingestion by both M types, indicating that both M
recognized the apoptotic thymocyte via its expression of PS. Although
flow cytometry disclosed differences in several receptors known to be
involved in clearance of apoptotic cells in other cell types, blockade
of a variety of such receptors did not indicate an explanation for the
deficiency of AM in phagocytosis of apoptotic cells. In addition to
explaining our previous observation that apoptotic lymphocytes are
detected with surprising ease in the lungs, these results have
important implications for the role of the lungs in development of
autoimmunity.
The observation that murine AM are markedly deficient in ability to
ingest apoptotic thymocytes emphasizes the importance of examining the
functional capacities of Mø in different organs. AM patrol one of
the body’s largest interfaces with the external environment, which
they must keep free of inhaled particles and aspirated microbes without
engendering excessive inflammation. The frequency of pneumonia, the
most common lethal infection in hospitalized adults, on one hand, and
the increasing frequency of asthma in industrialized nations on the
other hand, illustrates the narrow balance needed to maintain this
equilibrium. AM are a highly differentiated type of M that have a
predominantly suppressive role on the induction of immune responses
(35, 36); however, AM do permit local expression of T
cell effector functions while inhibiting their proliferation
(37). Although AM ultimately derive from the bone
marrow, there is considerable experimental evidence showing that steady
state numbers of resident AM derive largely from proliferation
within the lung (38, 39, 40). Defective ingestion of apoptotic
human neutrophils by resident rabbit AM has been shown previously
(12). Our results confirm and extend that finding to
murine AM and apoptotic lymphocytes (both thymocytes and a cloned T
cell line). In addition, the results of our in vivo experiments
demonstrate that this deficiency is not an artifact due to AM
removal from the unique environment of the lungs or of the in vitro
assay.
These results are also significant because they extend to AM
recognition of apoptotic cells by their surface expression of PS.
Because AM are normally exposed to pulmonary surfactant, which is
uniquely rich in PS, it was conceivable that they would eschew this
recognition pathway. Instead, our annexin-blocking experiments show a
major contribution of PS recognition, as has previously been shown for
a variety of other M types in humans and rodents
(14, 15, 16). The negative results we obtained in blocking
specific M integrins using both M types provide important data on
resident tissue M that have previously received little attention for
this function. Our results using resident murine AM and PM agree
with those of Platt and associates (41), who previously
showed that Abs against type 3 complement receptor (CD11b/CD18) or VNR
(CD51/CD61) did not block ingestion of apoptotic thymocytes by
thioglycolate-elicited murine PM. These results are in contrast to
the effect of blocking VNR by mAb or RGDS on ingestion of apoptotic
human neutrophils by HMDM and by rat bone marrow-derived monocytes
(BMDM), and of apoptotic murine thymocytes by the murine M cell line
J774 (16, 19, 42). As discussed below, we believe this
difference in results is chiefly due to the state of differentiation of
the M studied. The mAbs against integrins and the RGDS tetrapeptide
we used were in saturating concentrations and have been shown in other
systems to block receptor function. Nevertheless, we cannot exclude the
possibility that these Abs did not block an epitope specific for
recognition or phagocytosis of apoptotic cells. Moreover, the
relatively late stage apoptotic thymocytes used may be an additional
explanation for the lack of inhibition seen with anti-VNR mAbs. Our
results using rmC5-3 should not be interpreted to exclude unequivocally
a role for CD14 in ingestion of apoptotic lymphoid cells by resident
murine AM and PM, as this mAb has been shown to enhance rather
than block LPS-induced release of TNF- by J774 cells
(26). Indeed, only two anti-CD14 mAb among several
tested have been found to inhibit uptake of apoptotic cells
(18). Reagents to test additional receptors that have been
implicated in clearance of apoptotic cells in other model systems
(e.g., CD36, CD68) are not available in the mouse. Our results
underscore the multiplicity of receptors used by M to ingest
apoptotic cells and the likelihood that additional, uncharacterized
receptors exist.
Several models have been presented to explain the interactions of this
multiplicity of M receptors. Fadok, Savill, and colleagues
(14) proposed that the receptors used for recognition of
apoptotic targets depend primarily on the activation state of the M
rather than on its species or site of origin, or the specific apoptotic
target cell. This model is based on their observation that both HMDM
and murine BMDM use VNR, whereas elicited inflammatory M are
dependent on recognition of PS. An activated phenotype with use of PS
recognition could be induced in murine BMDM by exposure to digestible
particulates such as ß-1,3-glucan via endogenous TGF-ß elaboration
(43). Interpreted in this regard, our results imply that
even in the absence of inflammation, resident murine AM and PM
also use this inflammatory pathway rather than the vitronectin pathway,
either because of tissue differentiation or due to the burden of
inhaled particles they routinely ingest. It could be argued that the
initial adherence step used in our studies could have induced a switch
from the vitronectin pathway to the use of PS recognition. Activation
of some M functions by even brief adherence steps has been observed
previously (44). This possibility is supported by our
observation that surface expression of CD14 increased on PM (but
only slightly on AM) after overnight incubation, which implies some
degree of activation. However, the 12–16-h period of M incubation
we used was shorter than the 5–7 days in culture needed to mature HMDM
or murine BMDM. Moreover, our in vitro results for resident PM agree
closely with those of Licht and associates (21), who
examined resident PM of BALB/c mice and who completed their in vitro
assay within 7 h of M harvest. Hence, we believe it more likely
that resident AM and PM recognize apoptotic lymphocytes primarily
via PS expression in vivo. Additional experiments will be needed to
test this possibility. More recently it has been observed that annexin
V, unlike PS liposomes or analogues, blocks phagocytosis of apoptotic
lymphocytes by both unactivated murine M (MBMDM and J774 cells) and
elicited murine PM (28). These findings, which agree
with our results, have led Schlegel and associates (28) to
suggest that recognition of PS is a general feature in the recognition
of apoptotic lymphocytes by murine M. The molecular nature of M
receptors for PS remains undefined (reviewed in Ref. 17).
None of these considerations detract from our major finding, that
differences between resident murine M in maximal rate of
phagocytosis of apoptotic lymphocytes depend on their organ of
origin.
Impaired clearance of apoptotic lymphocytes from the lungs could
contribute to development of autoimmunity in susceptible individuals by
making available nucleosomes, which are highly immunogenic particles
now recognized as a major autoantigen in systemic lupus erythematosus
(2). Nucleosomes are macromolecular complexes that form
the basic units of chromatin. They are composed of eight core histones
(four homodimers of H2A, H2B, H3, and H4), two superhelical turns of
DNA, and a single encircling histone H1 molecule. Nucleosomes are
polyclonal B cell activators in vitro (45, 46) and are
recognized specifically by T cell clones (47).
Nucleosome-specific CD4+ T cells are identifiable
in the spleens of lupus-prone mice by 1 mo of age, before other
abnormalities develop (2). Because nucleosomes are formed
in vivo exclusively by endonuclease digestion of chromatin during
apoptosis, impaired clearance of apoptotic lymphocytes provides a
potential mechanism for breaking peripheral self-tolerance. The
extensive DNA fragmentation we have previously demonstrated in lung
lymphocytes during a pulmonary immune response to a noninfectious agent
(5) indicates that nucleosomes can be formed with ease in
the lungs of mice. Even greater degrees of lymphocyte apoptosis,
particularly of the greatly expanded CD8+ T cell
effector populations, occur during the resolution of viral infections
(48, 49). Several considerations suggest that the lungs
are a key site of clearance of apoptotic lymphocytes. Normal lungs
contain a large fraction of the body’s total lymphocytes, primarily as
single cells within alveolar capillaries and walls (50, 51). Lung lymphocytes are highly enriched for activated T cells
(27, 52, 53, 54), many of which appear not to ever leave the
lungs (55). Thus, the observed deficiency in phagocytosis
of apoptotic lymphocytes by AM is even more surprising.
The reason for the characteristic of AM is currently unknown. Given
the extensive kinetic studies we performed, including relatively short
time points both in vitro and in vivo, we have rejected the possibility
that AM actually ingested and digested apoptotic lymphocytes so
rapidly that they could not be detected. We have considered three
potential mechanisms. First, AM ingestion of apoptotic cells may be
down-regulated due to previous ingestion of digestible particles or
even apoptotic cells themselves. The latter possibility was recently
demonstrated in vitro using rat BMDM and apoptotic neutrophils
(42). That study showed that an initial round of
phagocytosis led subsequently (after 48 h) to decreased
phagocytosis of apoptotic neutrophils, but not of opsonized
erythrocytes, which would be recognized via Fc receptors. Such a
specific defect in phagocytosis of apoptotic targets is just what we
found. A second, teleological possibility is that a level of
indifference to apoptotic cells is an evolutionary adaptation that
preserves the capacity of AM to produce proinflammatory cytokines
for host defense of the lungs. Phagocytosis of apoptotic neutrophils
has been shown to inhibit actively and specifically the production by
HMDM of IL-1ß, IL-8, IL-10, GM-CSF, and TNF-, as well as
leukotriene C4 and thromboxane
B2 (56). This
effect is mediated by autocrine/paracrine elaboration of TGF-ß,
PGE2, and platelet-activating factor
(56). Uptake of apoptotic lymphocytes by murine peritoneal
M has recently been shown to favor the growth of the protozoan
pathogen Trypanosoma cruzi via mechanisms that depend on
PGs, TGF-ß, and polyamines (57). A third possibility is
that AM are relatively deficient in currently unknown receptors for
recognition or phagocytosis of apoptotic leukocytes. These
possibilities are not mutually exclusive, and multiple mechanisms could
underlie the phenotype we observed in murine AM.
Two possible limitations of the current study should be considered.
First, we used overnight (12–16 h) dexamethasone incubation for the
majority of experiments because it resulted in very uniform thymocyte
apoptosis, as evidenced by the annexin-V positivity of >95%. This
treatment is longer than the 4–6-h treatment used in many other
studies, and it resulted in a relatively late stage of apoptosis
denoted by the 42% propidium iodide staining we found. Based on the
experiments using 6-h dexamethasone treatment, CTLL-2 cells, and
neutrophils, we do not believe that late apoptosis of the thymocytes
alone explains the underlying deficiency by AM. However, the
duration of apoptosis should be considered in interpreting the
inhibition experiments. Second, we examined only resident (i.e.,
nonelicited) AM and PM from normal mice. It is clear that the
relative deficiency we have found in phagocytosis of apoptotic T cells
by resident M from normal mice can be overcome at the level of the
total lung mononuclear phagocyte population during lung infection or
inflammation, when large numbers of dying T cells and other leukocytes
must be cleared rapidly and specifically. Previous studies have, in
fact, shown that mononuclear phagocytes recovered during resolving
pneumonia have ingested apoptotic neutrophils (58, 59, 60).
The capacity to recognize and ingest apoptotic cells is lacking in
freshly isolated human blood monocytes (12, 19), but is
induced rapidly (4 h) in a dose-dependent fashion by GM-CSF,
TGF-ß, IFN-
, and IL-1ß (61). Physiological
modification could occur via changes in resident AM themselves, by
altered differentiation of recruited blood monocytes in the
inflammatory environment, or both. Thus, our findings are most relevant
to the noninflamed lungs.
In summary, we have demonstrated an unanticipated and pronounced
deficiency in phagocytosis of apoptotic lymphocytes by resident murine
AM. Additional experiments are needed to define whether this defect
exists in human AM and to establish whether the defect can be
overcome by AM activation.
Note added in proof.
Since submission of this manuscript, Fadok and colleagues have described a stereo-specific human receptor for PS (62).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jeffrey L. Curtis, Pulmonary and Critical Care Medicine Section (111G), Department of Veterans Affairs Medical Center, 2215 Fuller Road, Ann Arbor, MI 48105-2303.
3 Abbreviations used in this paper: M, macrophage; AM, alveolar M; BAL, bronchoalveolar lavage; BMDM, bone marrow-derived M; H&E, hematoxylin and eosin; HMDM, human monocyte-derived M; PM, peritoneal M; PS, phosphatidylserine; RGDS, arginine-glycine-aspartic acid-serine; RGES, arginine-glycine-glutamic acid-serine; VNR, vitronectin receptor.
Received for publication February 9, 2000. Accepted for publication May 23, 2000.
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, PM
, AM
, Control
conditions. 
), 1 mM
RGDS (
) for 30 min at 4°C. Next, without
washing, apoptotic thymocytes were added to the treated M
, PM