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Respiratory Medicine Unit, Department of Medicine (RIE), Rayne Laboratory, The University of Edinburgh Medical School, Edinburgh, U.K.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Potential control mechanisms for regulation of the recognition and
phagocytic pathways by which apoptotic cells are removed include
prolongation of myeloid cell functional longevity, such as phagocytic
and secretory responses by inflammatory mediators and growth factors
(10, 11, 12). Inhibition of the rate of apoptosis in these cell populations
at inflamed sites would serve a dual purpose of regulating the
potential for neutrophil-mediated damage at these sites and the numbers
of neutrophils that are available for recognition by macrophages.
Inflammatory mediators also act to regulate phagocytosis of apoptotic
cells by macrophages. Multiple receptor pathways are implicated in the
phagocytosis of apoptotic cells, although specific pathways may be
dominant in certain environmental conditions (13, 14). For human
monocyte-derived macrophages, we have proposed a model in which
macrophage 
vß3 and CD36 act in concert to
allow apoptotic cell recognition (7, 13). Modulation of receptor
expression during monocyte differentiation may determine the phagocytic
ability of macrophage populations at different anatomic sites.
Induction of vß3 occurs concomitantly with
the acquisition of the capacity to phagocytose apoptotic cells during
in vitro culture of monocytes (15). For murine macrophages, different
phenotypes may be induced by local microenvironmental stimuli. Bone
marrow-derived macrophages, in contrast to those isolated from the
peritoneal cavity which utilize a phosphatidylserine based pathway, use
an vß3 integrin-based recognition
mechanism (16). In addition, phagocytosis of ß-1,3-glucan particles
can induce a switch between these two pathways (17). There is also
clear evidence that macrophage phagocytic responses are also influenced
by integrin-mediated signal transduction pathways (18). Thus, control
of apoptotic cell removal may be achieved by regulation of macrophage
phagocytic potential.
In this study, we have investigated whether modulation of protein kinase activity had a regulatory role in the process of macrophage recognition of apoptotic cells. In particular, we have examined the effects of short term treatment of human monocyte-derived macrophages with PGs, which elevate intracellular cAMP. Our results suggest that specific inhibition of phagocytosis of apoptotic neutrophils following cAMP elevation may involve disassembly of cytoskeletal elements involved in cell-substratum interaction without inducing cell detachment. These data support a model in which the matrix microenvironment exerts control over the macrophage capacity for clearance of apoptotic cells. Thus, pharmacologic targeting of elements of the PKA pathway may represent a novel strategy for the modulation of macrophage capacity for the clearance of apoptotic cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Iscove’s DMEM, HBSS, and culture supplements were from Life Technologies, Paisley, U.K., and Falcon tissue culture plastic was from A. & J. Beveridge, Edinburgh, U.K. Rhodamine-phalloidin was obtained from Molecular Probes (Eugene, OR). Dextran T500 was from Pharmacia, Milton Keynes, U.K. Normal rabbit serum was from the Scottish Ab Production Unit, Lanarkshire, U.K. Rabbit anti-human erythrocyte membrane Ags and FITC-conjugated F(ab')2 fragments of goat anti-mouse Ig were from Dako (High Wycombe, U.K.), MAb all of IgG1 isotype, 23C6 (CD51/CD61 complex) and 13C2 (CD51), were a gift from Dr. Mike Horton, Imperial Cancer Research Fund, London, U.K. PM6/13 (CD61) was a gift from Dr. Mike Wilkinson, Royal College of Surgeons, London, U.K. FA6-152 (CD36) was selected from Ab panels obtained from the Vth International Workshop on Leukocyte Differentiation Ags. PGs were purchased from Cascade Biochem., Reading, U.K. All other chemicals were obtained from Sigma Chemical, Poole, U.K.
Cell isolation
Mononuclear cells and neutrophils were isolated from peripheral blood as described (4, 19). Freshly citrated blood was centrifuged at 400 x g for 20 min, and the platelet-rich plasma supernatant was used to prepare autologous serum by addition of calcium chloride (10 mM final concentration). Leukocytes were isolated after removal of erythrocytes by sedimentation using 6% (w/v) dextran T500 in saline by fractionation on a discontinuous gradient of isotonic Percoll solutions made in Ca2+/Mg2+-free PBS (CMF-PBS) Percoll concentrations of 49.5, 63, and 72.9% at 700 x g for 20 min. Mononuclear cells were aspirated from the 49.5%/63% interface and neutrophils from the 63%/72.9% interface and washed three times in HBSS before culture. Neutrophils were cultured at 4 x 106/ml in Iscove’s DMEM containing 10% autologous serum at 37°C in a 5% CO2 atmosphere for 18 to 24 h. Neutrophils used in the phagocytosis assay were >50% apoptotic as determined by morphologic appearance, CD16 "low" expression, annexin V positivity and >98% of cells retain membrane integrity as assessed by the vital dye trypan blue. Monocytes were enriched from the mononuclear cells by selectively attaching them to 24-well plates for 1 h at 37°C. The nonadherent lymphocytes were removed and the adherent monocytes were washed twice in PBS. Monocytes were then cultured for 5 to 7 days in Iscove’s DMEM containing 10% autologous serum at 37°C in a 5% CO2 atmosphere. Their maturation into macrophages was assessed by flow cytometry using a panel of myeloid-specific and activation markers including CD16 and CD51/CD61 (data not shown).
Assay for macrophage phagocytosis of apoptotic neutrophils
Phagocytosis of apoptotic neutrophils was assayed by minor modifications of previously described methods (4, 7). Neutrophils that had been cultured in vitro were washed once with Iscove’s DMEM and resuspended at 4 x 106 cells/ml in Iscove’s DMEM alone. Adherent macrophages were washed in Iscove’s DMEM before addition of 1 ml of the above neutrophil suspension to each well. After 30 min of incubation at 37°C, the wells were washed four times with ice-cold CMF-PBS, fixed in 2.5% glutaraldehyde/PBS for 10 min, and then stained for myeloperoxidase at 37°C using 0.1 mg/ml dimethoxybenzidine and 0.03% (v/v) hydrogen peroxide in PBS. The percentage of macrophages that had phagocytosed myeloperoxidase-positive apoptotic neutrophils was quantified microscopically by examination of randomly selected fields and counting at least 500 cells/well. Results from each experiment were expressed either as the mean percentage of phagocytic macrophages of triplicate wells or as the percentage phagocytosis relative to untreated controls.
Macrophage phagocytosis of IgG-opsonized erythrocytes
Erythrocytes were washed and resuspended at 2.5 x 107/ml in Iscove’s DMEM and incubated at 4°C with rabbit polyclonal anti-human erythrocyte Ab (1:100) for 30 min. Opsonized erythrocytes were then washed and resuspended at 1 x 106/ml in Iscove’s DMEM and incubated with macrophages at a macrophage-erythrocyte ratio of 1:2.
Flow cytometry and immunocytochemistry
Flow cytometry was performed as described previously (19, 20), with all incubations conducted on ice. Six-day-old monocyte-derived macrophages were detached from cell culture plates by vigorous pipetting after incubation on ice with PBS for 30 min. Macrophages (1 x 105) were washed with ice-cold PBS containing 0.2% (w/v) BSA and 0.1% (w/v) sodium azide and preincubated with 20% (v/v) normal rabbit serum to block "nonspecific" binding to Ig Fc receptors. Cells were then incubated with saturating concentrations of mAb for 30 min. After washing, cells were incubated with FITC-conjugated F(ab')2 goat anti-mouse Ig (1:25) for 30 min and washed twice before flow cytometric analysis using an EPICS Profile II flow cytometer (Coulter Electronics, Luton, U.K.) (20). For indirect immunofluorescence analysis, macrophages were resuspended at 1 x 106/ml of Iscove’s DMEM and 105/well added to multispot microscope slides and allowed to adhere by incubation for 1 h at 37°C. After rinsing with Iscove’s DMEM, cells were fixed in 1% formaldehyde in PBS for 10 min. Cells were permeabilized using 1% Triton X-100 in PBS for 5 min and then washed thoroughly in PBS. Slides were blocked in PBS containing 20% normal rabbit serum for 10 min before incubation with primary Ab optimally diluted in PBS containing normal rabbit serum. Bound primary Ab was detected using F(ab')2 FITC-conjugated rabbit anti-mouse Ig Ab. For visualization of actin, rhodamine-phalloidin (0.15 µM) was included. Slides were mounted in 50% glycerol in PBS and examined using an Olympus BH-2 microscope with a fluorescent lamp attachment.
Treatment of monocyte-derived macrophages with agents that elevate intracellular cAMP
Macrophages were washed once with Iscove’s DMEM and incubated for various times as stated in text at 37°C with dbcAMP,3 8-Br-cAMP, or PGs diluted in Iscove’s DMEM. For cAMP determinations, PGE2 was added in the presence of a 2.5 mM concentration of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). The treated cells were then washed once with Iscove’s DMEM before assessment of macrophage phagocytosis or immunocytochemistry.
Determination of cAMP concentration
Macrophages were cultured adherent to six-well tissue culture plates for 6 days as described above. Macrophages were washed once with Iscove’s DMEM and incubated with Iscove’s DMEM alone or Iscove’s DMEM containing 0.5 mM IBMX and 3 µM PGE2 for 0.5, 2, 5, 10, 15, or 45 min. The medium was then aspirate and 600 µl of ice-cold 0.5 M trichloroacetic acid were added. Macrophages were extracted on ice for 20 min and then scraped off, and material was transferred to Eppendorf tubes. The samples were then vortexed and centrifuged (10,000 x g, 5 min), and 500 µl of the supernatant were added to 125 µl of 10 mM EDTA (pH 7.0) and 500 µl of freshly prepared 1,1,2-trichlorotrifluoroethane-tri-n-octylamine (50:50, v/v) (21). After further vigorous mixing, the samples were centrifuged (10,000 x g, 2 min), and 500 µl of the aqueous layer were removed and neutralized with 100 µl of 6 M sodium hydrogen carbonate. Cyclic AMP measurements were made on triplicate 50-µl aliquots from each sample. Each tube contained 50 µl of buffer (50 mM Tris-HCl, 4 M EDTA, pH 7.5), 50 µl of sample, or standard containing known amounts of cAMP (doubling dilutions from 0.125 to 16 pmol with 250 pmol of cAMP used to define nonspecific binding) and 100 µl [3H]cAMP (88,000 dpm; DuPont (U.K.), Stevenage, U.K.). The reaction was initiated by addition of 150 µl-cAMP-binding protein (final concentration, 0.85 µg protein/ml) and incubated overnight at 4°C. The reaction was terminated by the addition of 250 µl of 0.5% activated charcoal. After 3 min, the samples were centrifuged (10,000 x g, 4 min) and 200 µl of the supernatant were transferred to 5-ml scintillation vials. Five milliliters of Flo-Scint IV scintillation fluid (Canberra Packard, Pangbourne, U.K.) were added to each vial, and the samples were analyzed on a liquid scintillation counter.
Analysis of results
Results are expressed as the mean ± SEM and n = number of independent experiments using macrophages from different donors. Results were analyzed by one-way analysis of variance followed by the Newman-Keuls procedure to determine significant differences between groups. p values < 0.05 were considered to be significant where * represents p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human monocyte-derived macrophages were pretreated with the
membrane-permeable analogue of cAMP, dbcAMP, to mimic elevation of
intracellular cAMP levels, and phagocytosis of apoptotic neutrophils
was assessed. When compared with preincubation in medium alone, 2 mM
dbcAMP decreased the percentage of monocyte-derived macrophages that
phagocytosed apoptotic neutrophils (Fig. 1
). The low proportion of phagocytic
macrophages following dbcAMP treatment made assessment of the effects
of dbcAMP on the number of apoptotic neutrophils phagocytosed per
macrophage (phagocytic index) difficult, particularly since under
control conditions the phagocytic index was 
1.4. In a series of
experiments, maximal effects of 2 mM dbcAMP treatment were apparent
following 15 min of pretreatment with no further inhibition observed
with longer incubation times (data not shown). All subsequent
experiments were therefore performed using 15-min pretreatment times.
Since elevation of cAMP might result in functional alterations
downstream of the receptors mediating macrophage recognition of
apoptotic neutrophils that would affect all phagocytic processes, we
assessed whether dbcAMP had similar effects on Fc
R-mediated
phagocytosis. In contrast to the observed inhibition of
monocyte-derived macrophage phagocytosis of apoptotic cells,
pretreatment of macrophages with dbcAMP did not inhibit recognition of
Ig-opsonized erythrocytes (Fig. 1). Thus, treatment of macrophages with
an activator of PKA does not nonspecifically inhibit all phagocytic
pathways. Furthermore, modulation of phagocytosis of apoptotic
neutrophils following cAMP elevation suggests that the potential of
monocyte-derived macrophages to phagocytose apoptotic cells can be
rapidly and dynamically regulated.
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Arachidonic acid metabolites of the PG series are key inflammatory
mediators that interact with specific cell surface receptors to
activate adenylate cyclase resulting in an elevation of intracellular
cAMP. We therefore assessed the effects of a number of different PGs
for their ability to inhibit monocyte-derived macrophage phagocytosis
of apoptotic neutrophils. Macrophages were pretreated with either
PGE2 (10 µM) or PGD2 (10 µM) for 15
min before testing their phagocytic ability. We also compared the
effects of elevation of cGMP using dbcGMP (2 mM) in comparison with the
positive controls, dbcAMP (2 mM) and 8 Br-cAMP (2 mM), as described
above. We found that all the PGs tested were capable of inhibition of
macrophage phagocytosis of apoptotic neutrophils (Fig. 2). In contrast to the effects of
elevation of intracellular cAMP, raising cGMP levels did not inhibit
monocyte-derived macrophage phagocytosis. These results suggest that
macrophage phagocytosis can be regulated by intracellular signals
and that the levels of intracellular cAMP may have a role in
determining macrophage capacity for phagocytosis of apoptotic
neutrophils.
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Since the above data suggest that macrophages express multiple
receptors for different PG species, we next determined the relative
potency and efficacy of PGE2 and PGD2 in
comparison with dbcAMP for inhibition of phagocytosis of apoptotic
neutrophils. Results shown in Figure 3
indicate that half-maximal inhibition (IC50) is observed at
0.5 mM for dbcAMP treatment. Maximal inhibition of macrophage
phagocytosis was observed following pretreatment with 10 µM
concentrations of both PGE2 and PGD2. However,
PGE2 (to IC50 1 µM) was much more potent
that PGD2 (10 µM inhibited phagocytosis only by
25%).
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To determine whether pretreatment of monocyte-derived macrophages
with PGs exerted inhibitory effects via elevation of cAMP, we next
measured intracellular concentrations of cAMP in macrophages following
treatment with PGE2 using a radioreceptor assay. As
shown in Figure 4, PGE2 acted
rapidly to elevate cAMP, increasing cAMP levels 10-fold after 5 min.
Interestingly, observed elevation was transient, returning to basal
levels within 30 min. In contrast, inhibition of phagocytosis of
apoptotic neutrophils following PGE2 treatment was
prolonged with inhibitory effects observed after 1 h (data not
shown), implying that elevation of cAMP initiates signaling pathways
that ultimately determine phagocytic responses.
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One possible mechanism for the rapid regulatory effects of cAMP elevation would be altered expression of surface receptors involved in macrophage recognition of apoptotic cells. However, flow cytometric analysis indicated that there was no significant alteration in surface expression of CD51, CD61, and the 61D3 Ag, as assessed by changes in mean fluorescence intensity, following dbcAMP pretreatment (data not shown), suggesting that the observed effects did not involve modulation of surface receptor expression. We therefore examined the effects of cAMP elevation on the distribution of receptors involved in the recognition process. Indirect immunofluorescence analysis of ß3 integrins and CD36 on macrophages pretreated with dbcAMP for 15 min revealed that the cellular distribution of CD36 was similar on untreated macrophages and those pretreated with dbcAMP. Although heterogeneity in terms of the pattern of distribution of the integrin ß3 subunit was observed in both untreated and treated macrophage populations and treatment of macrophages with dbcAMP resulted in an increase in the proportion of macrophages with a uniform ß3 distribution (data not shown), no clear association between ß3 distribution patterns and phagocytic capacity was apparent.
It has previously been demonstrated that elevation of cAMP induces
phenotypic alterations in macrophages and other cell types. Microscopic
examination of human monocyte-derived macrophages treated with dbcAMP
(Fig. 5, A and
B) and PGE2 (data not shown) revealed
clear morphologic alterations that were suggestive of altered
adhesion. However, quantitative analysis revealed that elevation of
cAMP did not alter the numbers of adherent macrophages (104 ± 4%
of adhesion of untreated macrophages, n = 3 separate
experiments). These data also demonstrate that observed inhibition of
phagocytosis is not due to selective detachment of phagocytic
macrophages. Moreover, additional experiments indicated that
pretreatment of macrophages with dbcAMP did not alter the ability of
macrophages to adhere to substrate (data not shown). Although dbcAMP
did not affect the percentage of adherent cells, when we examined the
intracellular distribution of cytoskeletal elements, we found that
colocalization of actin and talin into discrete structures
(representing contact points with substrate) was altered by dbcAMP
treatment (Fig. 5, C–F). Similar results
were found when we examined vinculin, paxillin, and tyrosine
phosphorylation patterns (data not shown), suggesting that dbcAMP
induces morphologic alterations by uncoupling adhesion receptors from
cytoplasmic cytoskeletal elements, including potential signal
transduction pathways (22).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we demonstrate that elevation of cAMP within
monocyte-derived macrophages specifically reduces macrophage potential
to recognize and phagocytose apoptotic neutrophils but not Ig-opsonized
erythrocytes, indicating that cAMP does not block all phagocytic
pathways in macrophages. Elevation of cAMP and activation of PKA may
signal rapid, subtle responses to microenvironmental stimuli, resulting
in reduced capacity for phagocytosis of apoptotic cells by macrophages.
The prostanoids PGE2 or PGD2 have
antiinflammatory effects on neutrophil function in terms of
granule secretion (24, 25, 26). However, our observation that macrophage
removal of apoptotic cells is inhibited by these agents raises the
possibility that they may increase the tissue load of apoptotic cells
at inflamed sites. Inflammatory mediators may therefore have complex
effects on processes involved in resolution of inflammation. One
possibility is that PG-induced cAMP elevation may provide
counterregulatory signals to proinflammatory mediators or CD44
cross-linking. Preliminary experiments indicate that dbcAMP treatment
inhibits CD44 augmented monocyte-derived macrophage phagocytosis of
apoptotic neutrophils (CD44 treated (20), 50.1 + 9% phagocytosis;
CD44 + 2 mM dbcAMP treated, 18.6 + 8%; n =
5). However, since dbcAMP also inhibits phagocytosis under control
conditions (see
Figs. 1–3
), it is difficult to determine the precise
relationship between cAMP elevation and CD44-mediated signals in
control of phagocytic capacity. We are currently further investigating
the effects of PGs and other antiinflammatory agents on macrophage
phagocytic responses in combination with GM-CSF or other
proinflammatory cytokines.
The observations reported here may be of great significance for removal of apoptotic neutrophils in situ. A variety of inflammatory mediators (including PGs) that stimulate adenylate cyclase activity and activate PKA (27) are likely to decrease apoptotic cell removal. All monocyte-derived macrophages may be capable of phagocytosis of apoptotic cells, providing that a certain activation "threshold" is reached, with PKA playing a regulatory role in limiting phagocytic responses. For instance, some monocyte-derived macrophage subpopulations, e.g., multinucleated giant cells, show low levels of phagocytosis of apoptotic cells but are readily able to phagocytose Ig-opsonized erythrocytes, suggesting that phagocytic competence is not the basis for the lack of apoptotic cell uptake. One possibility is that differential PKA activity in giant cells accounts for observed low level responsiveness to apoptotic cell stimuli.
Data presented in this article indicate that there are multiple levels of control for macrophage removal of apoptotic cells from inflamed sites. Although the mechanism by which cAMP elevation down-regulates macrophage phagocytosis of apoptotic cells remains to be determined, we have observed that there are rapid alterations in the distribution of cytoskeletal components (actin, talin, vinculin, and paxillin) in macrophages following dbcAMP treatment. These changes are accompanied by cell "rounding" when macrophages are examined by light microscopy and show parallels with the reported effects of PGs on murine macrophage adhesion, spreading, and motility (28, 29). Membrane ruffling and filopodia extension were inhibited by prolonged PG treatment in a reversible manner, resulting in altered macrophage adhesion. Together with recent evidence that in cytotoxic T cells PKA is able to phosphorylate Rho (30), a key regulator of cytoskeletal organization, these data indicate that intracellular cAMP levels may also have a pivotal role.
We have investigated the effects of agents that disrupt microfilament
and microtubule organization (cytochalasin B and nocodazole). Although
we did find inhibition of recognition of apoptotic cells when
macrophage were treated with nocodazole (2.5 µg/ml), it was difficult
to assess the effects of cytochalasin B (5 µg/ml) because the washing
procedure used for assessment of macrophage phagocytosis resulted in
significant detachment of cells in three of five experiments. These
findings suggest that treatment of macrophage with dbcAMP differs from
disruption of microfilaments in two ways. First, unlike cytochalasin B
treatment, dbcAMP does not inhibit phagocytosis of Ig-opsonized
erythrocytes (Fig. 1) (31, 32). Indeed, Newman et al. (32) have
previously demonstrated that cAMP does not inhibit FcR-mediated
phagocytosis. Second, although we have noted disassembly of structures
that may represent areas of contact with substrate and cell
"rounding," treatment of macrophages with dbcAMP does not result in
detachment, and cells remain firmly adherent through multiple washes
that are necessary for assessment of phagocytic capacity. Adhesion of
macrophages to extracellular matrix components, e.g., fibronectin, has
previously been shown to modulate C or Ig receptor-mediated
phagocytosis (33, 34), and it is tempting to speculate that the
extracellular matrix composition of inflamed sites exerts indirect
regulatory effects on the process of removal of apoptotic cells. One
possibility is that intracellular levels of cAMP are altered following
integrin-mediated adhesion to extracellular matrix components as found
for human neutrophils (35).
In summary, elevation of cAMP, using cell permeable analogues of cAMP (dbcAMP, 8-Br-cAMP) or by receptor-directed stimuli (PGE2, PGD2), in macrophages specifically inhibits phagocytosis of apoptotic cells but not Ig-opsonized erythrocytes. Modulation is independent of changes in the percentage of adherent macrophages, although a specific, rapid redistribution of cytoskeletal elements was observed, suggestive of changes in the adhesive status of macrophages. These data indicate that intracellular cAMP may have a key role in controlling the capacity for removal of apoptotic cells by macrophages at inflamed sites during the resolution of inflammatory processes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Adriano G. Rossi, Respiratory Medicine Unit, Department of Medicine (RIE), Rayne Laboratory, The University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 9AG Scotland, U.K. E-mail address:
3 Abbreviations used in this paper: dbcAMP, dibutyryl-cAMP; dbcGMP, dibutyryl-cAMP; IBMX, 3-isobutyl-1-methylxanthine; 8-Br-cAMP, 8-bromo-cAMP; PKA, protein kinase A.
Received for publication August 15, 1997. Accepted for publication December 9, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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v integrin expression on cultured human macrophages. Proc. Natl. Acad. Sci. USA 90:2517.RIII) expression. J. Immunol. 153:1254.[Abstract]
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 L.-P. Erwig, S. Gordon, G. M. Walsh, and A. J. Rees Previous Uptake of Apoptotic Neutrophils or Ligation of Integrin Receptors Downmodulates the Ability of Macrophages to Ingest Apoptotic Neutrophils Blood, February 15, 1999; 93(4): 1406 - 1412. [Abstract] [Full Text] [PDF]
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) or Ig-opsonized
erythrocytes (
). Relative percentage phagocytosis is expressed
compared with control medium pretreatment cells representing mean
± SEM of 14 separate experiments. In these experiments, the percentage
of cells ingesting apoptotic neutrophils under control conditions was
20 ± 4% and following dbcAMP treatment was 10 ± 3%. The
percentage of control macrophages ingesting opsonized erythrocytes was
30 ± 3%, whereas in dbcAMP-treated macrophages the value was
unchanged (31 ± 4%). *p < 0.05 compared with
control.
) PGE2 (•), and PGD2 (
