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vß3)1
Departments of
*
Medicine and
Pediatrics, National Jewish Medical and Research Center, and
Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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v, and up to 20% by
oxidized low density lipoprotein and
N-acetylglucosamine, implying a major role for integrin
and minor roles for scavenger and lectin receptors. Uptake into
macrophages stimulated with ß-1,3-glucan was blocked 50% by PS
liposomes and 40% by oxidized low density lipoprotein, suggesting that
the macrophages had switched from using integrin to recognition of PS.
MEM-18 and 61D3 (anti-CD14 mAbs) were poor inhibitors of apoptotic
neutrophil uptake, but good inhibitors of apoptotic lymphocyte uptake.
The switch to PS recognition was accompanied by down-regulation of
vß3 expression and function. Anti-CD36
blocked uptake into unstimulated or stimulated macrophages, suggesting
CD36 involvement not only with the vß3
integrin mechanism (as previously reported) but also with PS
recognition. A maximum of 70% inhibition was achieved by combining
anti-CD36 with either anti-av or PS
liposomes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages appear to recognize apoptotic cells via different
mechanisms, including integrin, phosphatidylserine
(PS)3 recognition, scavenger
receptors, and lectins. Human monocyte-derived macrophages (HMDM)
(4, 5, 6), human alveolar macrophages (7), and murine bone marrow-derived
macrophages (8) seem to phagocytose apoptotic cells via the
vß3 integrin system, which on human
macrophages associates with CD36. This complex then binds to secreted
thrombospondin (TSP), which binds to an undefined ligand on the
apoptotic cell (6). Elicited murine peritoneal macrophages, phorbol
ester-treated THP-1 cells, and glucan-stimulated mouse bone
marrow-derived macrophages have been shown to recognize exposed PS on
the surface of apoptotic cells (9, 10). Although the recognition is
stereospecific, the putative PS receptor (PSR) has not yet been
characterized. Third, macrophages may use one or more members of the
scavenger receptor family (SR) to engulf apoptotic cells. SRA-I/II on
murine thymic macrophages and elicited peritoneal macrophages (11),
SRB-1 on transfected CHO cells (12), CLA-1 on transfected HEK293 cells
(13), CD36 (6), and macrosialin/CD68 on resident mouse peritoneal
macrophages (14, 15, 16) all reportedly mediate binding and/or uptake of
apoptotic cells. Of these, SR-B1, CLA-1, CD36, and CD68 have been
reported to bind to PS- and phosphatidylinositol (PI)-containing
liposomes (13, 14, 15, 16, 17); however, it remains unclear whether they recognize
PS on apoptotic cells. Fourth, HMDM have been reported to phagocytose
apoptotic leukocytes using CD14, inhibited specifically by the mAbs
61D3 and MEM-18 (18, 19). How CD14 functions is not known. Some reports
differentiate it from the vß3/CD36/TSP
system (18) and the putative PSR (20), whereas Pradhan and co-workers
suggest that these are all part of a single, large receptor complex
(21). Lastly, mouse resident peritoneal macrophages have been shown to
use a lectin that is inhibited by N-acetylglucosamine (NAG)
(22).
Of these recognition systems, only the
vß3/CD36/TSP system and CD14 have been
demonstrated in HMDM uptake of apoptotic cells. Human macrophages have
been shown to recognize PS on sickled red cells or symmetric red cell
ghosts (23, 24, 25, 26); however, it has been suggested that they are unable to
recognize PS on apoptotic cells (4, 27, 28). We have shown that
recognition of PS on apoptotic cells by murine bone marrow-derived
macrophages can by induced after treatment with a digestible
particulate stimulus such as ß-glucan (10, 29); however, the lack of
an Ab to mouse CD36 prevented our assessment of its involvement. Our
objectives for this study were to determine whether PS recognition
could be induced in HMDM, what role, if any, CD36 played in PS
recognition, and what other receptors might be involved in phagocytosis
by either stimulated or unstimulated macrophages. For the purposes of
this study, we have defined PS recognition strictly as inhibition of
phagocytosis by PS-containing liposomes, although others have suggested
that inhibition by symmetric red cell ghosts in the absence of
inhibition by PS liposomes is also suggestive of PS recognition (21).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sterile plastic ware was purchased from Becton Dickinson
(Franklin Lakes, NJ) and Corning Costar (Cambridge, MA). DMEM, RPMI
1640, HBSS, and PBS were obtained from Life Technologies (Grand Island,
NY). X-Vivo 10 medium was obtained from BioWhittaker (Walkersville,
MD). FCS was purchased from Gemini Bioproducts (Calabasas, CA).
Acetylated low density lipoprotein (AcLDL), low density lipoprotein
(LDL), and oxidized low density lipoprotein (OxLDL) were obtained from
BTI (Stoughton, MA). Phosphatidic acid (PA), phosphatidylcholine (PC),
phosphatidylethanolamine (PE), PI, and PS were purchased from Avanti
Polar Lipids (Alabaster, AL). Sterile ß-1,3-glucan was purchased from
Accurate Chemical Co. (Westbury, NY). BSA, human serum albumin,
antibiotics, sterile 1.1-mm latex beads, dimethoxybenzidine
(o-dianisidine HCl), NAG, Arg-Gly-Glu-Ser (RGES),
Arg-Gly-Asp-Ser (RGDS), L--glycerophosphorylserine
(GPS), L--glycerophosphorylcholine,
phospho-L-serine, phospho-D-serine (PDS), EDTA,
and all remaining chemicals were purchased from Sigma (St. Louis, MO).
Antibodies
Monoclonal IgM mouse anti-human CD36 (clone CB38/NL07),
monoclonal IgM mouse anti-CD15 (clone H198), monoclonal IgM mouse
anti-CD15s (clone 2F3), monoclonal IgG1 mouse anti-human CD45,
CD61 (ß3), CD51/61(vß3)
CD64, CD16, and monoclonal IgG2b mouse anti-human Cd32 were
purchased from PharMingen (San Diego, CA); IgG1 anti-human
v derived from mouse ascites was obtained from Life
Technologies; and two monoclonal mouse anti-human CD68 Abs were
purchased from Cortex (IgG1; San Leandro, CA) and Monosan (IgM; Amuden,
Netherlands). The mAb 61D3 (IgG1 mouse anti-human) was a gift from
J. D. Capra and Drs. Andrew Devitt and Christopher Gregory. MEM-18
(mouse IgG1) was purchased from Monosan/Sanbio and TUK4 was purchased
from Caltag Laboratories (Burlingame, CA). Tetramethyl rhodamine
isothiocyanate- or Cy3-conjugated AffiniPure F(ab')2 of
goat anti-mouse IgG and IgM Abs were purchased from Jackson
ImmunoResearch Laboratories (West Grove, PA).
Human cell isolation and culture
Healthy unmedicated adult human subjects donated 400 ml of whole blood under a protocol approved by National Jewish Center’s institutional review board. Mononuclear cells were separated by dextran sedimentation and centrifugation through a discontinuous plasma-Percoll gradient as described previously (30). After three washes with HBSS, the cells were suspended at 4 x 106/ml in DMEM, and plated in 48-well tissue-culture plates at 0.5 ml/well. Following 1-h incubation at 37°C in 5% CO2, the cell layer was washed twice to remove nonadherent leukocytes, and the adherent monocytes were given X-Vivo 10 medium containing 10% (v/v) pooled human serum (heated at 65°C for 30 min) plus 100 U/ml penicillin and 100 mg/ml streptomycin (HMDM medium). The monocytes were cultured in 37°C in 5% CO2, with medium exchanged on days 3 and 7. Day 7–10 HMDM were used, according to preliminary studies for maximal macrophage uptake of apoptotic cells (data not shown).
Neutrophils were isolated from the modified dextran/plasma Percoll preparation of human blood as described above (30), washed twice with HBSS, and suspended at 5 x 106/ml in RPMI 1640 containing 10% (v/v) FCS. With this preparation, neutrophils were >98% viable and pure, as measured by trypan blue exclusion and histologic analysis.
Stimulation of HMDM
Day 5–8 HMDM were stimulated with 25 µg/ml ß-1,3-glucan or 5:1 latex beads, suspended in HMDM medium. After 48 h the monolayer was washed twice with HBSS to remove noningested particles before examination for recognition of apoptotic cells.
Neutrophil apoptosis
Neutrophils were placed in 75-cm2 tissue culture flasks, irradiated with a 302-nm UV transilluminator for 10 min, transferred to 50-ml plastic tubes, and rotated end-over-end in a 5% CO2 incubator at 37°C for 3 h. The cells were then washed twice and resuspended in RPMI plus 10% FCS at 1 x 108/ml for use in the phagocytosis assay. This treatment induced 77 ± 1.7% apoptosis by analysis of morphologic changes with <3% necrosis, as assessed microscopically for nuclear condensation and trypan blue exclusion (n = 24). These apoptotic neutrophils have been shown to express PS externally using annexin-V-FITC labeling and flow cytometry (31). For some experiments, apoptotic neutrophils were opsonized as previously described with mouse anti-CD45 and rabbit anti-mouse IgG (2).
Jurkat apoptosis
For some experiments, Jurkat T cells were used. They were routinely cultured in RPMI and 20% FCS containing L-glutamine, penicillin, and streptomycin. To induce apoptosis, Jurkat cells were irradiated as described for neutrophils.
Assay for macrophage phagocytosis of apoptotic cells
Uptake was determined as previously described (1, 32). Apoptotic
neutrophils were added to day 7–10 HMDM at a 5:1 ratio in HMDM medium
and incubated for 2 h at 37°C in 5% CO2 to achieve
maximal neutrophil uptake, based on preliminary time-course and
dose-response studies (data not shown). The monolayer was then washed
with PBS to remove attached noningested neutrophils, fixed with 1%
formalin, and treated with dimethoxybenzidine to stain for
myeloperoxidase (MPO) as a marker of the ingested neutrophils (1, 32).
The macrophages themselves were routinely negative for peroxidase
staining. Using x40 light microscopy, one investigator (M.L.W.)
examined the macrophages for uptake of apoptotic neutrophils, counting
two replicate wells each of 200 HMDM/well, and a second investigator
(V.A.F.) made blinded confirmation of representative plates. The
phagocytic index (
I) was calculated as the percentage of
phagocytosing macrophages x the average number of neutrophils
ingested per macrophage. Macrophages that showed discrete, round,
MPO-positive inclusion(s) were scored as having ingested one (or more)
apoptotic neutrophils, while HMDM that showed diffusely MPO-stained
cytoplasm, presumably due to earlier digestion of one or more
neutrophils, were scored as having ingested only one neutrophil. Those
neutrophils whose margins extended more than 50% beyond the edge of
the macrophage cell membrane were scored as noningested. (This
convention may have underestimated the number of phagocytosed
neutrophils, because ultrastuctural analyses have shown macrophages to
engulf such neutrophils with a thin wall of cytoplasm (32).) To verify
that the results were not specific for neutrophils, apoptotic Jurkat
cells were offered; phagocytosis was evaluated in the same way, except
that the MPO stain was not used.
Although the absolute value for the control phagocytic index
varied daily between HMDM preparations, within each experiment this
value was highly reproducible. To facilitate comparison of the results,
we report the phagocytic index in several experiments as the percentage
of the mean of the control phagocytic index replicates for that
experiment (normalized I).
Receptor inhibitors: ligands and Abs
To study HMDM use of the candidate receptors, the following
ligands, Abs, and controls were used: RGDS, RGES, anti-human
v, anti-human CD36, GPS, GPC, PLS, PDS, AcLDL,
OxLDL, LDL, anti-human CD68, and NAG. Unilamellar phospholipid
vesicles were prepared as previously described (9). PC liposomes were
made at 100 mol % PC, and PA, PE, PI, and PS liposomes were mixed at
70 mol % PC/30 mol % phospholipid. Macrophages were preincubated with
the receptor ligands, Abs, or their negative controls for 30 min before
adding apoptotic neutrophils. All inhibitors were examined for their
ability to inhibit phagocytosis nonspecifically by examining the uptake
of zymosan and apoptotic neutrophils opsonized with CD45 as previously
described (2). In no case were any of the inhibitors effective in
decreasing phagocytosis of either of these particles (data not shown).
As an additional control, opsonized neutrophils were fed to macrophages
in the presence or the absence of anti-CD64, anti-CD32, and
anti-CD16 (each at 50 µg/ml).
Down-regulation of CD36
HMDM were cultured in 100-mm bacteriologic plates to facilitate harvesting. They were still adherent, and preliminary experiments showed that they phagocytose apoptotic cells using mechanisms identical with those cultured on tissue culture plastic (not shown). Day 5 HMDM were stimulated with 25 µg/ml glucan or were left unstimulated. After 48 h macrophages were harvested by replacing the medium with ice-cold HBSS plus 10 mM EDTA and gently detaching cells with a cell scraper. HMDM were then washed twice with HBSS to remove EDTA and were resuspended in HBSS to 5 x 105/ml. Ninety-six-well, flat-bottom tissue culture plates were precoated for 2 h at 4°C with 50 µl of HBSS containing 100 µg/ml anti-CD36, 100 µg/ml human serum albumin, or 100 µg/ml anti-CD45. After rewarming the coated plates to 37°C, 50 µl of the HMDM suspension/well was adhered over 30 min at 37°C in 5% CO2 before adding apoptotic neutrophils for the phagocytosis assay. In select plates, HMDM were treated with 0.1 mmol of PS liposomes or 150 µg/ml OxLDL for 30 min before adding apoptotic cells.
Immunofluorescence labeling of surface receptors
HMDM were cultured in bacteriologic plates. Day 5 HMDM were stimulated with 25 µg/ml glucan or were left unstimulated. After 48 h, select plates were pretreated for 30 min with 0.1 mmol of PS liposomes or 150 µg/ml OxLDL. Macrophages were harvested as described above, added at 1 x 106/100 µl in HBSS to 96-well round-bottom tissue culture plates, and washed twice with chilled Krebs-Ringers phosphate buffer with dextrose plus 0.01% BSA. Primary Ab was added for a 30-min incubation on ice (anti-CD36, 100 µg/ml; 61D3, 12.5 µg/ml; anti-CD45, 100 µg/ml; anti-VnR: anti-CD61 or anti-CD51/61, and anti-CD68, all at 1/20 dilution). After three washes, the cells were incubated for 30 min on ice in the dark with 100 µg/ml of the relevant secondary Ab (tetramethyl rhodamine isothiocyanate- or Cy3-conjugated F(ab')2 goat anti-mouse IgG or IgM). HMDM were then washed a final three times and suspended at 1 x 106/ml in Krebs-Ringers phosphate buffer with dextrose plus 0.01% BSA. Samples were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, Sunnyvale, CA).
Statistical analysis
For phagocytosis studies, a mixed effects model was used to
analyze data for each inhibitor (33), with the response variable being
the absolute value of I. If the distribution of I indicated a
high degree of positive skewness, it was transformed using a natural
logarithm. The covariance structure of the repeated measurements was
evaluated by comparing Akaike’s information criterion between models,
specifying several commonly occurring covariance structures (34). In
every case, we found the data to conform to either a compound symmetric
or an unstructured covariance pattern. Pairwise multiple comparisons
between conditions were made using the Tukey-Kramer multiple
comparisons procedure at the 0.05 level of significance. All analyses
were performed in SAS using the Mixed procedure (35). Although
statistical analysis was conducted on the absolute or log-transformed
value of the I, we reported the results using normalized I to
facilitate comparison.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3/CD36/TSP and PS
recognition mechanisms
To confirm use of the
vß3(VnR)/CD36/TSP mechanism in
phagocytosing apoptotic neutrophils and to test for a potential switch
to the PS recognition mechanism, we examined the function of these
receptors in control, glucan-stimulated, and latex-stimulated
macrophages. The tetrapeptide RGDS was used to block integrin
(vß3) binding, and PS liposomes were used
to bind the putative PSR, with RGES and PC liposomes as negative
controls. In unstimulated HMDM, pretreatment with RGDS, but not RGES
(not shown), blocked phagocytosis of apoptotic neutrophils by
approximately 50% (Fig. 1
). Increasing
the concentration of RGDS to 10 mM reduced phagocytosis no further
(data not shown). Neither PS nor PC liposomes were active in inhibiting
apoptotic cell uptake. Latex treatment did not change this inhibition
pattern (Fig. 1). In contrast, glucan treatment was associated with
loss of inhibition by RGDS and acquisition of inhibition by PS
liposomes (Fig. 1). Testing concentrations from 5–75 µg/ml, we found
25 µg/ml glucan to maximize both the macrophage’s capacity for
phagocytosis and the generation of PS-inhibitable activity (data not
shown). This concentration was therefore used in subsequent
experiments, although it should be noted that this level is not
achievable in mycotic infections in vivo. After testing this
concentration for 24, 48, and 72 h, we found that PS-inhibitable
uptake was maximal following 48 h of treatment, so this time point
was chosen for all additional experiments. Inhibition of apoptotic cell
uptake by PS liposomes was likewise dependent on the concentration of
total phospholipid. Concentrations of PS liposomes from 0.001–0.1 mM
caused progressive inhibition of apoptotic cell uptake into stimulated
HMDM, while higher concentrations had no further effect (not shown). In
contrast, we found that opsonized apoptotic cells were taken up by
nearly all the macrophages; the mean percent positive macrophages for
unstimulated cells was 86.7 ± 7.8%, and that for stimulated
cells was 91.2 ± 6.3%. For both populations of cells, 90%
inhibition of uptake was achieved using a combination of mAbs against
Fc receptors (anti-CD64, anti-CD32, and anti-CD16; data not
shown).
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). To confirm recognition of
the polar head group of PS, we examined the inhibitory action of
water-soluble phosphoester derivatives of PS using GPS and PLS, with
GPC and PDS as negative controls (Fig. 3). Again, in glucan-stimulated HMDM, PS
liposomes markedly reduced macrophage ingestion of apoptotic
neutrophils, while PC liposomes did not. Similarly, GPS and PLS blocked
uptake, while GPC and PDS were inactive. These results imply that
stimulated HMDM did not simply interact with apoptotic neutrophils
through their negative charge or hydrophobicity, but that they
specifically recognized PS on the surface of the apoptotic cell. None
of the inhibitors had any effect on the uptake of zymosan or opsonized
apoptotic cells.
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vß3/CD36/TSP mechanism as previously
reported by Savill and co-workers (6), but glucan stimulation promoted
a switch to use of the proposed PSR. Further, the PSR accounted for a
similar portion of apoptotic cell uptake into glucan-stimulated
macrophages (
50%), as did the
vß3/CD36/TSP mechanism into unstimulated
HMDM.
The next step was to use Abs to vß3 and
CD36 to determine their ability to inhibit phagocytosis of apoptotic
cells by unstimulated and glucan-stimulated HMDM. RGDS and PS liposomes
were used to demonstrate the ability of the macrophages to switch to
the PSR. As expected, in unstimulated HMDM, anti-CD36,
anti-v, and RGDS reduced phagocytosis by 30–40%,
while PS liposomes had no inhibitory effect (Fig. 4). Following glucan stimulation, there
was loss of RGDS and anti-v inhibition, but gain of
PS inhibition. Anti-CD36 blocked phagocytosis by both. Other IgM Abs
against surface Ags (CD15, expressed at low levels, and CD15s,
expressed at high levels, determined by flow cytometry) were also used
at the same concentrations; these had no effect on the uptake of
apoptotic cells (data not shown). These findings argued that CD36 was
required for phagocytosis of apoptotic cells by both resting and
stimulated HMDM.
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vß3 and CD36 on unstimulated
and stimulated macrophages was determined by flow cytometry. We found
that 58.6 ± 11.9% (range, 37.2–70.0%) of unstimulated
macrophages were CD36 positive. After glucan stimulation, the
percentage of CD36-expressing macrophages was unchanged at 58.9 ±
10.6% (range, 43.6–76.2%; n = 7). We also observed
no change in CD45 expression. Using anti-CD51/61 and anti-CD61
Abs to detect vß3, we found a shift in
fluorescence in all the cells that was not seen when the isotype
control was used, suggesting that all the macrophages expressed this
VnR at low levels. Despite low expression on unstimulated cells, we
observed a statistically significant decrease in expression on
glucan-stimulated macrophages (Fig. 5).
This decrease may account in part for why this Ab no longer inhibits
phagocytosis by stimulated cells; however, it may be accompanied by
decreased function of the vß3 receptor, as
we suggested for mouse macrophages (10)
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It was striking to observe that despite the mechanism used, only 50% inhibition was achieved, suggesting that other mechanisms were contributing to uptake of apoptotic cells. We sought to clarify this issue by examining the inhibition of other receptors reported to mediate phagocytosis of apoptotic cells, including SRs other than CD36, CD14 (recognized by the mAb 61D3), and lectins (11, 12, 13, 14, 15, 16, 17, 18, 19, 22).
Scavenger receptors
SRs are a rapidly enlarging receptor class recognizing different
lipoprotein derivatives. SRA-I/II bind AcLDL and OxLDL, but not LDL or
PS (35, 36); the structurally related MARCO receptor (which has
expression restricted to splenic and lymph node macrophages) likewise
binds AcLDL (37, 38). Members of the B class of SRs, SR-B1 and the
human homologue CLA-1, CD36, and macrosialin (CD68), an OxLDL receptor,
exhibit different binding affinities for these lipoproteins, but all
appear capable of binding PS and PI at least when offered in a liposome
(13, 15, 16). SR-B1 and CLA-1 reportedly bind all three lipoproteins
(3, 37, 39). CD36 binds OxLDL and not LDL (40); its binding of AcLDL is
controversial (37, 40). CD68 binds OxLDL alone (41, 42, 43). To test the
roles of these various SR in apoptotic cell uptake, we pretreated HMDM
with 150 µg/ml of AcLDL, OxLDL, or LDL. In our system, these
concentrations did not appear to be toxic, as the macrophages could
still phagocytose zymosan and opsonized apoptotic cells (not shown). In
unstimulated HMDM, OxLDL, but not AcLDL or LDL, inhibited phagocytosis
by approximately 20%. In glucan-stimulated HMDM, the inhibitory effect
of OxLDL was more pronounced, reducing phagocytosis by almost 40%,
while AcLDL and LDL were again inactive (Fig. 6). Subsequent dose-response studies,
using up to 250 µg/ml OxLDL did not further inhibit phagocytosis in
either unstimulated or stimulated macrophages (data not shown).
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CD14
Another reported mechanism for HMDM phagocytosis of apoptotic
cells is the 61D3-inhibitable receptor CD14 (18, 19, 45), which might
function separately (18, 20) or jointly with the integrin and
phospholipid receptors (21). Using the 61D3 mAb, we achieved
inconsistent results; in some experiments we observed 20% inhibition
of apoptotic neutrophil uptake, and in some experiments we observed no
inhibition. This may have been due to donor variation or differences in
the preparations of 61D3 that we used, although we observed no
differences in the ability of these preparations to bind to macrophages
as detected by flow cytometry. We therefore purchased a second
anti-CD14 Ab (MEM-18), which was shown by Devitt and colleagues to
inhibit the binding of apoptotic lymphocytes to CD14 (19). Levels of
CD14 expression detected by MEM-18 were similar to those detected by
61D3; 38% of cells from either unstimulated or stimulated macrophage
populations cultured for 7 days were positive (data not shown). We then
compared the effects of MEM-18 and TUK-4 (an anti-CD14 Ab that does
not inhibit the interaction of apoptotic cells with macrophages (19))
on the phagocytosis of apoptotic neutrophils and lymphocytes
(irradiated Jurkat T cells). The results of these experiments are shown
in Fig. 7. Although MEM-18 was effective
in reducing the uptake of apoptotic Jurkat T cells by both unstimulated
and stimulated macrophages (p < 0.003), it was
not effective in inhibiting neutrophil uptake. Taken together with the
data we collected using 61D3, these results suggest that CD14 mediates
the binding and uptake of apoptotic lymphocytes, but that it is of
minor importance in mediating the binding or phagocytosis of apoptotic
neutrophils.
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Lastly, to determine whether HMDM used a lectin-like receptor to phagocytose apoptotic neutrophils, we examined their ingestion of apoptotic cells in the presence of NAG. In unstimulated macrophages, NAG pretreatment reduced the phagocytosis of apoptotic cells by approximately 20% (not shown); this difference was statistically significant (p < 0.04; n = 12) and differs from the previous findings of Savill et al. (46). Increasing the NAG dose to as high as 40 mM inhibited phagocytosis no further (data not shown). In glucan-stimulated HMDM, NAG had no significant effect. Despite the statistical significance, it seems unlikely that lectins contribute in a major way to phagocytosis by human macrophages, and, in fact, the effect of NAG may be nonspecific.
Use of receptors in combination
After observing the inhibitors’ individual effects on HMDM
phagocytosis of apoptotic cells, it became important to determine
whether combining inhibitors would give additive effects. Using RGDS to
block the vß3/CD36/TSP mechanism, PS
liposomes to inhibit the PSR, OxLDL to block SR (presumably CD36), and
NAG to inhibit a possible lectin-like receptor, we pretreated
unstimulated and stimulated HMDM with the following combinations before
addition of apoptotic neutrophils: NAG and OxLDL; NAG and RGDS; RGDS
and OxLDL; NAG, OxLDL, and RGDS; and PS and OxLDL. No inhibitor
or combination of inhibitors reduced phagocytosis much below 50% of
control levels, and we observed no additive effects (Fig. 8). Using the combination of
anti-v and anti-CD36 in unstimulated
macrophages, we achieved a maximal inhibition of 68.3 ± 3.2%
(n = 6); the addition of 61D3 to this combination
showed a slight, but not statistically significant, additive effect
(74.3 ± 5.5%). Using the combination of anti-CD36 and PS
liposomes in stimulated cells, we achieved a maximal inhibition of
70.8 ± 4.5% (n = 6); adding 61D3 to this
combination gave 75.3 ± 3.7% inhibition. The effects of MEM-18
were the same as those of 61D3 (not shown).
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Because CD36 appeared to be required for the uptake of apoptotic
cells into both unstimulated and glucan-stimulated HMDM and because PS
liposomes and OxLDL failed to inhibit uptake additively into stimulated
HMDM, we questioned whether the putative PS receptor we have proposed
might be CD36. In fact, CD36 has been reported to recognize PS because
binding of AcLDL to CD36-transfected COS cells can be competed with PS
liposomes (17). In addition, Ryeom et al. have shown that CD36 is
expressed on retinal pigment epithelium that phagocytoses photoreceptor
outer segments in a PS-inhibitable fashion (47). However, PI liposomes
are equally or more effective in both these systems, and we did not
observe inhibition of phagocytosis by PI liposomes (see Fig. 2
).
Nevertheless, we hypothesized that if CD36 was able to recognize PS on
apoptotic cells, binding of its Ab would be inhibited by PS liposomes.
This is not an unreasonable hypothesis, as Ramprasad et al. have shown
that binding of Abs to CD68 (OxLDLR) can inhibit OxLDL binding to THP-1
cells and vice versa (15). After harvesting, macrophages were
pretreated with either PS liposomes or OxLDL before addition of
anti-CD36 Ab and were examined by flow cytometry. Following PS
liposome pretreatment, the percentage of CD36-positive HMDM remained
unchanged in both unstimulated and stimulated macrophages
(CD36-positive macrophages (unstimulated HMDM, 57.4 ± 15.7%;
unstimulated HMDM and PS liposomes, 59.4 ± 15.7%;
glucan-stimulated HMDM, 61.5 ± 13.5%; glucan-stimulated HMDM and
PS liposomes, 62.7 ± 8.38%; n = 4). Similarly,
following OxLDL pretreatment, unstimulated and stimulated HMDM did not
change their expression of CD36 (CD36-positive macrophages: control
HMDM, 60.2%; control HMDM and OxLDL, 61.7%; glucan-stimulated HMDM,
62.23%; glucan-stimulated HMDM and OxLDL, 65.8%; n =
2). We next attempted to down-regulate CD36 expression by adding PS
liposomes or OxLDL for 30 min before harvesting the macrophages for
flow cytometry. CD36 expression was not effected by either pretreatment
(data not shown). These data implied that CD36 was not the PS receptor;
however, it was possible that the epitope to which the Ab bound was not
the epitope that bound to the apoptotic cell. This interpretation seems
less likely given that these Abs did inhibit phagocytosis of
PS-expressing apoptotic cells. We therefore hoped to clarify this issue
by down-regulating the expression of CD36 with anti-CD36 before
phagocytosis.
We plated macrophages on anti-CD36-coated dishes before assaying the phagocytosis of apoptotic neutrophils in the presence or the absence of PS liposomes or OxLDL. Anti-CD45 was used as the control. Unstimulated macrophages plated on anti-CD36 showed the expected reduction in uptake of apoptotic cells (55.2 ± 3%; n = 6); neither PS liposomes nor OxLDL decreased phagocytosis further (data not shown). Stimulated macrophages plated on anti-CD36 also had reduced apoptotic cell uptake (39.5 ± 10%; n = 6); OxLDL failed to inhibit phagocytosis further, and PS inhibited uptake by an additional 10%, which was not statistically significant (data not shown). Macrophages adhered to plates coated with human serum albumin or anti-CD45 Ab, as nonspecific controls, showed no change in phagocytosis, and the patterns of inhibition by RGDS and PS were consistent with whether the cells were stimulated. Our interpretation of these data is that CD36 is a necessary cofactor for phagocytosis by PS-recognizing macrophages, but that it may not necessarily function as a PSR.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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vß3/CD36/thrombospondin system described
by Savill and co-workers (6). In contrast, HMDM that had been
stimulated with glucan lost the ability to use this recognition process
but acquired the ability to recognize PS. Previous investigators have
examined human macrophages for use of the proposed PS receptor and were
unable to identify it (4, 27, 28). However, those studies were with
unstimulated macrophages, which, as confirmed here, do not use this
recognition system. CD36 appeared to function in both groups of
macrophages (25–40 and 40–55% uptake, respectively), suggesting a
role in both recognition mechanisms. CD14 played a minor role, if any,
in the uptake of apoptotic neutrophils by either unstimulated or
stimulated macrophages, although selected Abs against this Ag inhibited
the uptake of apoptotic lymphocytes. Our findings agree with those of
Flora and colleagues, who have shown that the CD14 mechanism appears to
be most efficient for some lymphocytes and that uptake of apoptotic
neutrophils or peripheral blood neutrophils is inhibited poorly by 61D3
(18). At this point, it is unclear whether the CD14 mechanism shows any
connection or cooperativity with the
vß3/CD36/TSP or the PSR mechanisms, but
the fact that macrophages that fail to use CD14 still recognize PS on
apoptotic neutrophils suggests that CD14 is not the PSR we have
proposed. We could not find evidence for the use of SRs other than
CD36; however, identification of new SRs remains an active area of
investigation. In both stimulated and unstimulated macrophages, a large
proportion of the uptake (30–50%) was not blocked by any of the
inhibitors we used, suggesting that additional mechanisms remain
uncharacterized.
The nature of the receptor(s) that mediate PS recognition remains
unclear. The binding of AcLDL to CD36 can be competed by PS liposomes,
suggesting that CD36 can bind PS directly (17) and may itself be a PSR
capable of both recognition and initiation of apoptotic cell uptake. In
fact, Ryeom et al. have shown that CD36 on retinal pigment epithelium
can bind and internalize PS-containing liposomes, and that
anti-CD36 and PS liposomes can inhibit the uptake of PS-expressing
photoreceptor outer segments (47). However, PI liposomes function
equally well or better in both these systems, and we have been unable
to demonstrate inhibition of phagocytosis using this anionic
phospholipid or AcLDL on either human (see Fig. 2) or mouse macrophages
(9). Although we found that down-regulation of CD36 was associated with
a loss of PS inhibition of stimulated macrophages, the binding of
anti-CD36 was not inhibited by PS liposomes, and CD36 was not
down-modulated by PS liposomes. These data suggest that CD36 may act as
a necessary cofactor for either the VnR system (for which the ligand
has not been defined) or PS recognition (for which the receptor has not
been defined). It is highly likely that there may be multiple PSRs,
some of which remain to be identified. In our hands, PS recognition is
specific, in that phagocytosis is inhibited stereospecifically by PS
and its analogues, GPS and phosphoserine. Other anionic phospholipids
have no effect, arguing against any of the currently identified class B
SRs mediating this function.
It is also highly likely that the ligands on apoptotic cells are complex. We have used an operational definition for the PSR we have proposed, which is inhibition of phagocytosis by PS liposomes. Pradhan et al. have shown that inhibition of phagocytosis by unstimulated macrophages can be inhibited by symmetric red cell ghosts and have suggested that this implies PS recognition. They therefore hypothesize that murine macrophages use multiple, partly overlapping systems to recognize apoptotic cells (21). In our hands, however, symmetric red cells and sickled red cells, like PS liposomes, only inhibited phagocytosis of stimulated macrophages and had no effect on unstimulated macrophages (unpublished data). If human macrophages recognize PS on symmetric ghosts and sickled red cells (23, 24, 25, 26), why do they only appear to see it on apoptotic cells after they are stimulated? It is possible that PS on a symmetric red cell is not equivalent to that on an apoptotic cell. Data supporting this have been provided by Terpstra et al., who showed that binding of macrophages to oxidized red cells was calcium dependent, whereas binding to apoptotic cells was not (48). We also found that binding or phagocytosis of apoptotic cells by PS-recognizing macrophages is calcium independent (data not shown). These data lend support to the idea proposed by Pradhan et al. that apoptotic ligands are complex structures.
In summary, then, PS recognition of apoptotic cells appears to be an inducible function in both human and mouse macrophages. CD36 seems to be an important cofactor for at least two different recognition mechanisms: that mediated by the VnR and that mediated by PSR. Why are there so many different receptor systems for apoptotic cells? One could speculate a number of reasons. First, removal of apoptotic cells may be so critical to normal tissue structure and function that multiple mechanisms have been developed. Second, one of the critical functions of the apoptotic cell when phagocytosed is to actively suppress macrophage proinflammatory functions, which may be mediated by one or more of these receptor mechanisms (2, 3). Third, there may be variations in ligand expression among different types of apoptotic cells. Support for this is provided by the work of Flora et al. (18) and Hart et al. (49). The former showed that the CD14 recognition mechanism appeared to be more effective for removal of lymphocytes than neutrophils, which our data confirm; the latter showed that CD44 treatment of human macrophages enhanced phagocytosis of apoptotic neutrophils but not lymphocytes. However, in most studies as well as those described herein there is a significant proportion of the recognition and uptake that is not blocked by any of the inhibitors identified to date, either alone or in combination. The clear implication is that a major mechanism for recognition and uptake of apoptotic cells remains to be identified.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Valerie A. Fadok, National Jewish Medical and Research Center, D509, 1400 Jackson St., Denver, CO 80206. E-mail address:
3 Abbreviations used in this paper: PS, phosphatidylserine; HMDM, human monocyte-derived macrophages; TSP, thrombospondin; PSR, phosphatidylserine receptor; SR, scavenger receptor; PI, phosphatidylinositol; NAG, N-acetylglucosamine; AcLDL, acetylated low density lipoprotein; LDL, low density lipoprotein; OxLDL, oxidized low density lipoprotein; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; RGES, Arg-Gly-Glu-Ser; RGDS, Arg-Gly-Asp-Ser; GPS, L--glycerophosphorylserine; GPC, L--glycerophosphorylcholine; PLS, phospho-L-serine; PDS, phospho-D-serine; MPO, myeloperoxidase; I, phagocytic index; VnR, vitronectin receptor.
Received for publication January 21, 1998. Accepted for publication July 27, 1998.
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W. Zou, J. Borvak, F. Marches, S. Wei, P. Galanaud, D. Emilie, and T. J. Curiel Macrophage-Derived Dendritic Cells Have Strong Th1-Polarizing Potential Mediated by {beta}-Chemokines Rather Than IL-12 J. Immunol., October 15, 2000; 165(8): 4388 - 4396. [Abstract] [Full Text] [PDF]
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 V. Terpstra, E. S. van Amersfoort, A. G. van Velzen, J. Kuiper, and T. J. C. van Berkel Hepatic and Extrahepatic Scavenger Receptors : Function in Relation to Disease Arterioscler Thromb Vasc Biol, August 1, 2000; 20(8): 1860 - 1872. [Full Text] [PDF]
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 G. H.-F. Chang, N. M. Barbaro, and R. O. Pieper Phosphatidylserine-dependent phagocytosis of apoptotic glioma cells by normal human microglia, astrocytes, and glioma cells Neuro-oncol, July 1, 2000; 2(3): 174 - 183. [Abstract] [PDF]
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U. A. Hirt, F. Gantner, and M. Leist Phagocytosis of Nonapoptotic Cells Dying by Caspase- Independent Mechanisms J. Immunol., June 15, 2000; 164(12): 6520 - 6529. [Abstract] [Full Text] [PDF]
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L. Temesvari, L. Zhang, B. Fodera, K.-P. Janssen, M. Schleicher, and J. A. Cardelli Inactivation of lmpA, Encoding a LIMPII-related Endosomal Protein, Suppresses the Internalization and Endosomal Trafficking Defects in Profilin-null Mutants Mol. Biol. Cell, June 1, 2000; 11(6): 2019 - 2031. [Abstract] [Full Text]
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N. Platt, H. Suzuki, T. Kodama, and S. Gordon Apoptotic Thymocyte Clearance in Scavenger Receptor Class A-Deficient Mice Is Apparently Normal J. Immunol., May 1, 2000; 164(9): 4861 - 4867. [Abstract] [Full Text] [PDF]
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 E. SELVI, S. MANGANELLI, R. DE STEFANO, E. FRATI, and R. MARCOLONGO CD36 and CD14 immunoreactivity of Reiter cells in inflammatory synovial fluids Ann Rheum Dis, May 1, 2000; 59(5): 399 - 400. [Full Text]
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C. Godson, S. Mitchell, K. Harvey, N. A. Petasis, N. Hogg, and H. R. Brady Cutting Edge: Lipoxins Rapidly Stimulate Nonphlogistic Phagocytosis of Apoptotic Neutrophils by Monocyte-Derived Macrophages J. Immunol., February 15, 2000; 164(4): 1663 - 1667. [Abstract] [Full Text] [PDF]
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L. Krishnan, C. J. Dicaire, G. B. Patel, and G. D. Sprott Archaeosome Vaccine Adjuvants Induce Strong Humoral, Cell-Mediated, and Memory Responses: Comparison to Conventional Liposomes and Alum Infect. Immun., January 1, 2000; 68(1): 54 - 63. [Abstract] [Full Text] [PDF]
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M.-K. Chang, C. Bergmark, A. Laurila, S. Horkko, K.-H. Han, P. Friedman, E. A. Dennis, and J. L. Witztum Monoclonal antibodies against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: Evidence that oxidation-specific epitopes mediate macrophage recognition PNAS, May 25, 1999; 96(11): 6353 - 6358. [Abstract] [Full Text] [PDF]
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S. C. Frasch, P. M. Henson, J. M. Kailey, D. A. Richter, M. S. Janes, V. A. Fadok, and D. L. Bratton Regulation of Phospholipid Scramblase Activity during Apoptosis and Cell Activation by Protein Kinase Cdelta J. Biol. Chem., July 21, 2000; 275(30): 23065 - 23073. [Abstract] [Full Text] [PDF]
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V. A. Fadok, A. de Cathelineau, D. L. Daleke, P. M. Henson, and D. L. Bratton Loss of Phospholipid Asymmetry and Surface Exposure of Phosphatidylserine Is Required for Phagocytosis of Apoptotic Cells by Macrophages and Fibroblasts J. Biol. Chem., January 5, 2001; 276(2): 1071 - 1077. [Abstract] [Full Text] [PDF]
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R. F. Thorne, J. F. Marshall, D. R. Shafren, P. G. Gibson, I. R. Hart, and G. F. Burns The Integrins alpha 3beta 1 and alpha 6beta 1 Physically and Functionally Associate with CD36 in Human Melanoma Cells. REQUIREMENT FOR THE EXTRACELLULAR DOMAIN OF CD36 J. Biol. Chem., November 3, 2000; 275(45): 35264 - 35275. [Abstract] [Full Text] [PDF]
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T. Miyahara, L. Schrum, R. Rippe, S. Xiong, H. F. Yee Jr., K. Motomura, F. A. Anania, T. M. Willson, and H. Tsukamoto Peroxisome Proliferator-activated Receptors and Hepatic Stellate Cell Activation J. Biol. Chem., November 10, 2000; 275(46): 35715 - 35722. [Abstract] [Full Text] [PDF]
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