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IMMUNOBIOLOGY
From the CRC Molecular Biology Group, Paterson
Institute for Cancer Research, Christie Hospital NHS Trust, Withington,
Manchester, UK.
Glycosaminoglycans (GAG) are a group of negatively charged
molecules that have been shown to bind and directly regulate the bioactivity of growth factors and cytokines such as basic fibroblast growth factor, transforming growth factor- Interleukin-10 (IL-10) is an 18-kd glycoprotein
produced by various cell types, including macrophages, activated T and
B lymphocytes, B-cell lymphomas, keratinocytes, and mast
cells.1-4 IL-10 was initially described as a novel
cytokine produced by murine Th-2 cellular clones that inhibited the
secretion of cytokines by Th-1 clones, notably interferon (IFN)-
Glycosaminoglycans are a group of linear polysaccharides consisting of
repeating disaccharide subunits in which one residue is an amino sugar,
either D-glucosamine (GlcN) or D-galactosamine (GalN), and the other either a hexuronic acid (D-glucuronic
acid [GlcA] or L-iduronic acid [IdoA]) or galactose.
They can be divided into 4 subgroups: heparan sulfate (HS) and heparin
(H), keratan sulfate (KS), chondroitin sulfates (CS; chondroitin
4-sulfate or chondroitin 6-sulfate) and dermatan sulfate (DS), and
hyaluronic acid (HA). With the exception of HA, which is made as a free
GAG and lacks sulfate, they are all synthesized as covalent complexes with core proteins, forming proteoglycans (PG), and are highly charged
because of the addition of sulfates at various
positions.22-26
GAG and PG are found in all tissue types as components of the
extracellular matrix and the basement and cellular membranes and in the
secretory granules of many cell types.22,24 At
inflammatory sites, soluble PG are secreted by activated leukocytes,
such as monocytes/macrophages, natural killer cells, T cells, mast
cells, and basophils, and they are released as a consequence of
extracellular matrix degradation.27-34 Besides their
prominent role as structural components, GAG, PG, or both have been
shown to modulate the activity of a number of cytokines by several
different mechanisms.35,36 For example, binding to
cell-surface PG, such as syndecans, or to the extracellular matrix PG
may provide tissue-bound reservoirs of cytokines that can be presented
to target cells, as has been previously described for basic FGF (bFGF)
and hematopoietic growth factors such as IL-3 and granulocyte
macrophage-colony-stimulating factor.37-39 Binding of
IL-7, IFN- In this study, we have identified hIL-10 as a heparin-binding cytokine.
The physiological relevance of hIL-10 binding to heparin or other GAG
can be expected to depend on the affinity of the cytokine for the GAG.
Accordingly, we have measured the affinity of hIL-10 for heparin and
investigated the biologic consequences of the interaction of hIL-10
with different types of GAG.
Cytokines and reagents
Binding of hIL-10 to heparin-agarose column
Solid-phase assay for binding hIL-10 to biotinylated albumin-heparin The binding of hIL-10 to immobilized heparin-albumin (biotinylated) was monitored using an IAsys auto plus device (Affinity Sensors, Cambridge, UK), as described by Lyon et al56 with some modifications. A planar biotinylated surface was derivatized with avidin and then loaded with the biotinylated albumin-heparin according to the manufacturer's instructions. All hIL-10 binding reactions were performed in 20 mmol/L Tris-HCl, pH 7.4, at 25°C, and data were collected at 0.3-second intervals. The association of hIL-10 with the immobilized heparin was monitored until a plateau was reached. The cuvette was then rapidly washed 4 times with 20 mmol/L Tris-HCl, pH 7.4, and the dissociation of hIL-10 from immobilized heparin-albumin was followed. The heparin-albumin surface was regenerated with 1 mol/L NaCl in 20 mmol/L Tris-HCl, pH 7.4, for 2 minutes and re-equilibrated with 20 mmol/L Tris-HCl, pH 7.4. Three independent sets of binding reactions were obtained in duplicate for 4 different hIL-10 concentrations (13.5 nmol/L-54 nmol/L). The association rate (ka) and the dissociation rate (kd) constants were calculated from a plot of the observed on rates (kon) and the hIL-10 concentrations at which they were carried out. The dissociation equilibrium constant (Kd) for hIL-10 binding to heparin-albumin was calculated using the ka and kd values as described above, where Kd = kd (y-axis intercept)/ka (gradient).Control of endotoxin levels All cell culture reagents used were either certified as low in endotoxin when purchased or were ensured to be low in endotoxin by the Pyrogenet-5000 turbidimetric LAL assay57 (BioWhittaker) using the Kinetic-QCL reader and WinKQCL software (BioWhittaker).Isolation of mononuclear cells from peripheral blood Peripheral blood from healthy donors was collected into tubes containing preservative-free heparin (CP Pharmaceuticals, Wrexham, UK) and processed within 1 hour of withdrawal. Mononuclear cells were isolated using a modification of the method described by Boyum.58 Peripheral blood was diluted 1:2.5 in phosphate-buffered saline (PBS), and 30 mL was then layered onto 15 mL Ficoll-Paque Plus (Pharmacia) and centrifuged at 400g for 35 minutes at 18°C. Mononuclear cells accumulating at the interface between the separating medium and the plasma were carefully transferred with a Pasteur pipette and washed twice with HEPES (25 mmol/L)-buffered RPMI 1640 medium (Gibco/BRL) supplemented with 10% fetal calf serum. Cell viability was determined by Trypan blue exclusion and found to be 95% to 98%.Cytokine and GAG treatment of peripheral blood mononuclear cells Peripheral blood mononuclear cells (PBMC) were suspended at a density of 2 × 106 cells/mL in HEPES (25 mmol/L)-buffered RPMI 1640 supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin, 2 mmol/L L-glutamine, and 10% FCS (all from Gibco). PBMC suspension (0.1 mL) was transferred to each well of a 96-well U-bottomed tissue culture plate (Nunc). Human IL-10 (10 ng/mL) or IFN- (10 ng/mL) were preincubated with various
concentrations of GAG for 20 minutes at room temperature in medium
without FCS. Subsequently, 0.1 mL was added to the PBMC-containing
96-well plates in duplicate and incubated for 24 to 72 hours at 37°C
in a humidified atmosphere containing 5% CO2. After the
incubation period, cells were pelleted by centrifugation at
200g for 3 minutes followed by resuspension in 200 µL PBS.
Cell-surface levels of CD16 or CD64 on monocytes/macrophages were
determined by direct immunofluorescence and analyzed by flow cytometry.
Immunofluorescence staining of cells for FACS analysis Harvested PBMC were washed twice with PBS, 2% bovine serum albumin, and 0.02% sodium azide (FACS buffer) by centrifugation and resuspended in 50 µL ice-cold FACS buffer containing mouse-antihuman CD16, or CD64 followed by incubation in the dark at 4°C for 30 minutes. Subsequently, the cells were washed 3 times with FACS buffer and resuspended in 200 µL PBS containing 1% formaldehyde. After a further incubation period of 1 hour on ice, the cells were washed and resuspended in 200 µL FACS buffer before analysis. Immunostained cells were analyzed on a FACScan flow cytometer (Becton Dickinson) using the CELLQuest software. Binding of antibodies was evaluated on monocyte/macrophage populations, which had been gated within PBMC by forward- and side-scatter patterns; the cells excluded from analysis were smaller and comprised less than 5% of the cell population. Monocyte/macrophage enrichment to more than 95% by the chosen flow cytometry gating was routinely confirmed by the expression of the monocyte-specific marker, CD14, of an identically gated control cell suspension. At least 5000 events gated for the forward- and side-scatter parameters characteristic of monocytes/macrophages were acquired. Results are expressed either as mean fluorescence intensity or percentage of maximum response within the gated region.Inhibition of proteoglycan sulfation by sodium chlorate Chlorate ions are potent inhibitors of GAG sulfation.59,60 Sodium chlorate (NaClO3) was used to test whether abrogation of GAG sulfation modified the mitogenic activity of hIL-10. PBMC, 2 × 105 cells, in 96-well U-bottomed tissue culture plates were incubated for 24 hours in the presence or absence of NaClO3 (5 or 20 mmol/L) in sulfate-free Dulbecco minimal essential medium (DMEM) containing 2 mmol/L L-glutamine, 2% FCS, 0.8 mmol/L magnesium chloride, and 1 mmol/L sodium pyruvate. Penicillin and streptomycin were omitted because they contain sulfur, and cysteine and methionine were reduced to 20% of normal concentration because they could also provide exogenous sulfate, which could negate the effect of chlorate. Ionic concentration was maintained at 0.15 mol/L by adjustment of NaCl concentrations. After 24 hours, cells were washed once and resuspended in culture medium containing hIL-10 (10 ng/mL) and chlorate. NaClO3 remained present at all times during the stimulation period. An equivalent concentration of NaCl + 1 mmol/L magnesium sulfate (MgSO4; sulfate-reconstituted medium) was used as the control. In some experiments, 15 mmol/L MgSO4 was added with hIL-10 to reverse the effect of chlorate. Cultured cells were recovered 48 hours after stimulation with hIL-10, and the expression of CD16 was determined by immunofluorescence staining and analyzed by flow cytometry.
Binding of hIL-10 to heparin agarose The ability of hIL-10 to bind to heparin agarose was investigated by chromatography of a semipurified preparation of hIL-10, also containing MBP, and factor Xa. The heparin-bound proteins were eluted using a stepwise salt gradient. From the stained gels it can be seen that MBP (42 kd) has little or no interaction with heparin-agarose, whereas both hIL-10 (18 kd) and factor Xa (30 kd) bind (Figure 1B). Factor Xa typically eluted from the column at 300 mmol/L NaCl, hIL-10 had stronger affinity, with peak levels of protein eluting at 600 mmol/L NaCl.Kinetics of hIL-10 binding to sensor chip-immobilized heparin The IAsys technology, which uses an evanescent field to measure changes in refractive index when a soluble analyte (here, hIL-10) binds to an immobilized ligand (heparin-albumin), has been previously used to investigate the binding kinetics of hepatocyte growth factor/scatter factor to DS.56 Using this technology we investigated the kinetics of hIL-10 binding to immobilized heparin-albumin. Biotinylated albumin-heparin was immobilized on an avidin-activated sensor chip, at a density of 293 arc seconds (1 arc second = 1/3600 of 1°). When hIL-10 was injected over the heparin-albumin surface, a typical sensogram was obtained (Figure 2), with an association phase (A), an equilibrium phase (E), and, when hIL-10 was replaced by running buffer alone, a dissociation phase (D). The association phase of the binding reaction between hIL-10 and heparin-albumin was fast, reaching saturation within 120 seconds (Figure 2A). Observed on rates (kon) were plotted against the hIL-10 concentration to calculate the association rate (ka, gradient) and dissociation rate (kd, y-axis intercept) constants (Figure 2B). The mean association rate (ka) was 3.5 × 105 mol/L 1 S1 (Figure
2B). The dissociation phase was considerably slower, with less than
25% of the bound hIL-10 dissociating from heparin after 5 minutes of
washing with buffer (Figure 2A). The mean dissociation constant
(kd) was 0.019 S1 (Figure 2b).
Association kinetics pointed to the existence of a single
heparin-binding site on hIL-10. Combined data from 3 sets of
interactions gave a calculated equilibrium dissociation constant,
Kd of 54 (± 7) nmol/L.
Soluble heparin and heparan sulfate inhibit hIL-10 activity Based on our results showing that hIL-10 binds to heparin agarose at physiological pH, we have postulated that heparin or other GAG may participate in regulating the cytokine activity. Human IL-10 has been previously shown to up-regulate the expression of CD64 on purified monocytes12 and CD16 on macrophages within PBMCs.61 Initial experiments were carried out to determine the optimal concentration of hIL-10 required for the induction of CD16 and CD64 on monocytes/macrophages within PBMC. Figure 3 shows that hIL-10 up-regulates the expression of both CD64 (Figure 3A) and CD16 (Figure 3B) on monocytes/macrophages in a concentration-dependent manner at 72 hours. Optimal concentrations of hIL-10 for CD16 and CD64 induction were found to be between 1 and 10 ng/mL, which corresponds to 2.47- to 4.68- and 2.95 to 6.25-fold increases in the levels of CD16 and CD64 expression, respectively. Similar percentage increases were seen at days 1 and 2; however, the background levels, which increased with time, were lower at day 1 (data not shown). Subsequently, similar experiments were carried out to investigate the effects of soluble GAG on hIL-10 activity using the optimal concentration of hIL-10 (10 ng/mL). Figure 4 shows that heparin and heparan sulfate markedly reduce (by up to 95%) the hIL-10-induced expression of CD64 (Figure 4A,C) and CD16 (Figure 4B,D) on monocytes/macrophages in a concentration-dependent manner at 24 and 72 hours. The 50% inhibitory concentrations (IC50) for heparin and heparan sulfate were found to be between 100 and 500 µg/mL on all days tested.
Like hIL-10, IFN- Soluble C-4S and DS, but not C-6S, inhibit hIL 10-activity The ability of other structurally distinct GAG, such as C-4S, DS, and C-6S, to regulate the activity of hIL-10 was also investigated. Preliminary experiments indicated that C-4S, DS, and C-6S had a much lower ability than heparin or heparan sulfate to modulate hIL-10 activity (data not shown). As a result, subsequent experiments were carried out using GAG concentrations ranging from 10 to 5000 µg/mL. Figure 5 shows that the biologic activity of hIL-10 on monocytes/macrophages, as determined by the induction of CD16 and CD64, were inhibited by soluble C-4S and DS. The addition of these GAGs at 5000 µg/mL resulted in a 60% to 75% reduction in the levels of CD64 and CD16 expression on hIL-10-treated monocytes/macrophages. C-6S, on the other hand, was shown to have a considerably lower capacity to inhibit hIL-10 function because it reduced the expression of CD16 and CD64 only by approximately 25% and 10%, respectively. The IC50 values for C-4S and DS (2000-2500 µg/mL) were considerably higher than those of heparin and heparan sulfate (100-500 µg/mL).
N-sulfate dependence of heparin binding to hIL-10 To examine the relative importance of different sulfate groups in heparin for the binding of hIL-10, specifically desulfated heparins were tested for their ability to inhibit the hIL-10-induced expression of CD16 on monocytes/macrophages. Figure 6 shows that N-acetyl-de-O-sulfated heparin, which lacks both N- and O-sulfate groups, had little or no effect compared to unmodified heparin (IC50 value of approximately 100 µg/mL) on the hIL-10-induced expression of CD16. Similarly, the replacement of N-sulfate groups with N-acetyl groups (de-N-sulfated heparin) resulted in a modest (approximately 20%) reduction in hIL-10 activity when used at 1000 µg/mL. When high concentrations were used, O-sulfate groups appeared partially to compensate for the absence of N-sulfates; a 60% reduction in hIL-10 activity was seen at 5000 µg/mL. Removal of O-sulfate groups had little or no effect on the inhibitory activity of heparin. Similar results were also obtained for CD64 (data not shown). Taken together, our results indicate that sulfate groups, specifically N-sulfates, are essential in the binding of heparin to hIL-10.
Sodium chlorate treatment of PBMC reduces activity of hIL-10 on monocytes/macrophages Our results suggest that the sulfation of exogenously added heparin is important in enabling it to inhibit the activity of hIL-10. Thus, we addressed the question of whether sulfation of cell-bound HS or CS/DS PG is required for hIL-10 to modulate CD16 expression. Sodium chlorate has been shown by several investigators to inhibit the sulfation of proteoglycans in intact cells.59,60 Figure 7A shows that treatment of PBMC with 20 mmol/L NaClO3 reduced the ability of hIL-10 to up-regulate the expression of CD16 by approximately 60%. However, this inhibition could be reversed by the simultaneous addition of 15 mmol/L MgSO4 to the culture medium. No significant effect on hIL-10 activity was seen when 5 mmol/L NaClO3 was used. In the absence of exogenous hIL-10, chlorate or MgSO4 treatment had no effect on the constitutive levels of CD16 (data not shown). In addition, we investigated the influence of chlorate on the capacity of TNF- to up-regulate the expression of ICAM-1 molecules
on monocytes/macrophages.63 Soluble heparin does not
affect the ability of TNF- to up-regulate the expression of ICAM-1
on human umbilical vein endothelial cells,49 an
observation that is consistent with its inability to bind to immobilized heparin under physiological conditions.62
Therefore, chlorate should not affect its mitogenic activity. Figure 7B
shows that the stimulation of PBMC with TNF- (20 ng/mL) alone
resulted in a 1.7-fold increase in the levels of ICAM-1 on
monocytes/macrophages compared to untreated cells. In contrast to
hIL-10, treatment of PBMC with chlorate did not inhibit the mitogenic
activity of TNF-. These data indicate that sulfate groups of PG are
required for the full mitogenic activity of hIL-10 on
monocytes/macrophages within PBMC.
Numerous growth factors and cytokines bind to heparin or heparan
sulfate molecules.64,65 These include the FGF
family,66 hepatocyte growth factor (HGF),67
chemokine platelet factor 4 (PF4),68 and cytokines such as
IL-7,40 IL-8,69 IL-12,70 and
IFN- We have demonstrated by affinity chromatography that hIL-10 displays a
strong affinity for heparin at pH 7.4. Human IL-10 could be eluted from
the column with 0.3 to 0.6 mol/L NaCl. This compares with 0.3 to 0.6 mol/L for IL-7,40 0.4 to 1.2 mol/L for
TGF- To test our hypothesis, we used an assay based on that described by te
Velde et al12 and Olikowsky et al,61 where
hIL-10 was shown to up-regulate the expression of CD64 on purified
monocytes and CD16 on macrophages within PBMC. In our experiments we
looked at the monocyte/macrophage (CD14+ cells) population
within PBMC rather than purified cells. The main advantage of the PBMC
system is that it allows critical intercellular contacts to take place
and, hence, is more like an in vivo situation. In this system hIL-10
was shown to up-regulate the expression of CD16 and CD64 on
monocytes/macrophages within PBMC in a concentration-dependent manner;
optimal concentrations of hIL-10 were between 1 and 10 ng/mL (Figure
3). Preincubation of hIL-10 with heparin or heparan sulfate reduced its
ability to up-regulate the expression of CD16 and CD64 on
monocytes/macrophages by up to 95% (Figure 4). The inhibitory effects
of heparin and heparan sulfate were shown to be concentration
dependent, with IC50 values ranging from 100 to 500 µg/mL. Heparin is the most negatively charged GAG (the average
disaccharide contains 2.7 sulfate groups) and usually binds more
strongly to ligands than do other GAGs. Although heparan sulfate is
biosynthetically related to heparin, it contains an average of only one
sulfate or less (depending on the source) per disaccharide. In general,
it contains more GlcNAc and GlcA, whereas heparin has a high IdoA/GlcA
ratio and predominantly contains GlcNSO3.22-26
Another feature of heparan sulfates that makes their distinction
possible from heparin is a chain structure consisting of alternating
low-sulfated domains (N-acetylated; NA domains) and domains
with high sulfation levels (N- and O-sulfated;
S-domains). The levels of sulfation within these S-domains can approach
the levels seen in heparin.22-26 Thus, the similar
IC50 values exhibited by these 2 GAG in our experimental
system may be accounted for by the presence of S-domains in heparan
sulfate, and they may reflect a requirement for a specific, highly
sulfated sequence for hIL-10 binding and not the overall negative
charge. Consistent with our findings, S-domains have been implicated in
the binding of heparan sulfate to IL-8,21
PF4,68 TGF As for the requirement for anionic groups on GAG for the binding of
hIL-10, we have clearly demonstrated the absolute requirement for
sulfation because N-acetyl-de-O-sulfated heparin
failed to inhibit the activity of hIL-10 compared to unmodified
heparin. Additionally, the relative lack of effect of the solely
O-sulfated GAGs, CS and DS, coupled with the marked
reduction in the inhibitory activity of heparin after the removal of
N-sulfates but not O-sulfates, indicates a strong
specific requirement for N-sulfation in heparin binding to
hIL-10. It must be noted, however, that at very high heparin
concentrations, O-sulfate groups were able partially to compensate for the lack of N-sulfates. Even among the
O-sulfated GAG, there seems to be some degree of
selectivity; the 4-O-sulfated DS and C-4S were able to
inhibit partially the biologic activity of hIL-10 at very high
concentrations, whereas the 6-O-sulfated C-6S had little or
no effect. The selective requirement for N-sulfates in the
binding of hIL-10 by heparin described here is comparable to that seen
for TGF The antagonistic effects of GAG on hIL-10 function described here are
consistent with the findings of several other studies in which soluble
GAG have been shown to abrogate the biologic activity of cytokines such
as IL-740,46 and IFN- Interestingly, whereas soluble GAG were shown to inhibit hIL-10
function, we could demonstrate that the inhibition of endogenous PG
sulfation with sodium chlorate59,60,82 reduced the
hIL-10-mediated expression of CD16 on monocyte/macrophages by up to
60%. This effect was shown to result from the specific depletion of
sulfation on PG because PBMC treated simultaneously with both chlorate
and sufficient exogenous sulfate to counter the effect of the chlorate retained normal responsiveness to hIL-10. Furthermore, the mitogenic activity of TNF- Several investigators have shown that low concentrations of soluble heparin can overcome the inhibitory effects of chlorate on cellular responses to bFGF.86,87 We have examined the effects of exogenously-added heparin or heparan sulfate at low concentrations (0.1, 1, and 10 µg/mL) on chlorate-treated cells and found them to be unable to restore the full activity of hIL-10 (data not shown). As proposed for HGF,83,88 our findings imply that though soluble GAG can modulate the activity of hIL-10, they cannot replace the function of cell-surface-associated PG. Although heparin and heparan sulfate are structurally similar, their
distribution in vivo is radically different. Heparin is synthesized in
connective tissue-type mast cells and is released in a soluble form on
degranulation.22 Heparan sulfate, on the other hand, is
found ubiquitously in an immobilized form in extracellular matrices,
basement membranes, and cell surfaces.89 These different distributions suggest different functions with respect to hIL-10 binding. Thus, through its interaction with hIL-10, heparin may antagonize its activity by competing with membrane hIL-10R, or it may
act as a carrier molecule protecting it from protease degradation and
increasing its half-life, as has been shown for IL-740 and IFN- The ability of GAG to interact with hIL-10 adds an additional layer of control to its biologic activities and may have important ramifications for the development of therapeutic agents in clinical situations in which hIL-10 has been implicated. For example, hIL-10 has been shown to inhibit macrophage-mediated immunity against intracellular and extracellular parasites, such as fungal and mycobacterial infections (eg Leishmaniasis, leprosy, and tuberculosis). In addition, hIL-10 is thought to act as an autocrine growth factor or inhibitor of antitumor immune responses in certain cancers, such as Burkitt lymphoma, Hodgkin disease, nasopharyngeal carcinoma, and AIDS-associated B lymphomas. Hence, our findings showing that GAG can bind to and inhibit the activity of hIL-10, coupled with the apparent requirement for specific N-sulfates, patterns of O-sulfation residues, and length of the sulfated segments, suggest possible clinical applications of chemically modified heparinoids as hIL-10 antagonists with low anticoagulant activity, alone or in combination with other therapies. In view of this, further studies are required to identify the minimum structural motifs for GAG binding to hIL-10.
The authors thank Dr M. Lyon and Prof J. Gallagher for review of the manuscript and helpful discussion, Mike Hughes and Jeff Barry for their technical assistance with flow cytometry analysis, and Jane Bolton for performing the lipopolysaccharide assays.
Submitted October 14, 1999; accepted May 12, 2000.
Supported by the Cancer Research Campaign, London, UK.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Shahram Salek-Ardakani, CRC Section of Molecular Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Withington, Manchester, M20 4BX, UK; e-mail ssalek{at}picr.man.ac.uk.
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Top
Abstract
Introduction
, IL-7, and
interferon-
such as fibroblast growth factor (FGF), transforming growth factor (TGF)-
LRRCHR-(105-110), which is
similar to one consensus site, XBBXBX (where B represents a cationic
lysine, arginine, or histidine residue and X represents a hydropathic
residue) and is described by several investigators
) or heparan sulfate (
) for 24 hours (A,B) or 72 hours
(C,D). CD64 and CD16 expression on monocytes/macrophages were
determined as described in Figure 
), DS (
), or C-6S
(
) for 72 hours. After the incubation period, CD16
expression on monocytes/macrophages was determined as described in
Figure 