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Originally published online as doi:10.1189/jlb.0504270 on August 17, 2004

Published online before print August 17, 2004
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(Journal of Leukocyte Biology. 2004;76:1066-1074.)
© 2004 by Society for Leukocyte Biology

Scavenger receptor A mediates H2O2 production and suppression of IL-12 release in murine macrophages

Szczepan Józefowski1 and Lester Kobzik

Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts

1 Correspondence: Harvard School of Public Health, 665 Huntington Avenue, SPH-2, Room 223, Boston, MA 02115. E-mail: sjozefow{at}hsph.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although class A type I/II scavenger receptor (SR-A) is involved in numerous macrophage functions, its signaling ability remains uncertain. We used monoclonal antibodies (mAb) to specifically stimulate receptors on mouse alveolar (AMs) and peritoneal macrophages (PMs). Immobilized anti-SR-A (2F8) and anti-Fc{gamma}R II/III (2.4G2) mAb stimulated hydrogen peroxide (H2O2) production in normal C3H/HeJ AMs (by 55% and 98%, respectively) and resident PMs (66% and 128%). The 2F8 mAb-stimulated H2O2 production resulted from specific stimulation of SR-A, since this response was absent in AMs from SR-A-deficient or C57BL/6 mice—the latter strain expressing an allelic form of SR-A, unrecognizable by 2F8 mAb. H2O2 production stimulated by anti-SR-A but not by anti-Fc{gamma}RII/III mAb was preserved in Fc{gamma}RI/III-deficient mice, ruling out involvement of Fc{gamma}Rs in the 2F8 mAb effect. In comparison with the Fc{gamma}R-stimulated respiratory burst, the response to anti-SR-A mAb was delayed and, unlike the former, inhibited by pertussis toxin. Ligation of SR-A also inhibited lipopolysaccharide plus interferon-{gamma}-stimulated interleukin-12 (IL-12) release, by 25% in AMs and by 68% in thioglycollate-elicited PMs, consistent with different levels of SR-A expression. Neither nitrite nor IL-6 accumulation was affected by anti-SR-A mAb. SR-A-stimulated H2O2 does not seem to mediate the inhibition of IL-12 release, since the inhibition was neither reversed by scavenging of H2O2 nor mimicked by exogenous H2O2. Our results indicate that SR-A not only mediates endocytosis but can also generate signals such as H2O2, which may affect microbicidal or proinflammatory functions.

Key Words: Fc{gamma} receptors • knockout • alveolar • peritoneal • hydrogen peroxide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Type I and II class A scavenger receptors are splice variants of a single gene product (referred to herein as scavenger receptor A1–2, SR-A). These SR-As are homotrimeric, helically coiled, integral membrane glycoproteins, containing SR cysteine-rich (missing in SR-A2) and collagenous domains in their extracellular portions [1 2 ]. SR-A is expressed predominately on macrophages and dendritic cells [3 4 ], furnishing these cells with the ability to bind a multiplicity of polyanionic ligands, including certain native, denatured, or covalently modified proteins, proteoglycans, polynucleotides, acidic phospholipids, sulfatides, sulfated polysaccharides, and bacterial components, such as lipopolysaccharide (LPS) and lipoteichoic acid [5 6 7 ]. Thanks to its extraordinary broad ligand-binding capacity, SR-A may participate in numerous macrophage functions. SR-A has been implicated in endocytosis of covalently modified proteins [8 9 ], in opsonin-independent phagocytosis of bacteria [6 10 11 12 ] and apoptotic cells [13 ], as well as in the clearance of anionic environmental particles from the lung [14 ] and LPS from the circulation [15 16 ]. SR-A is also involved in intercellular cooperation during antigen presentation [4 ] and in macrophage adhesion to the components of extracellular matrix [3 7 17 18 ] to other cells [19 ] and to tissue-culture plastic-adsorbed serum [20 ].

Since the identification of SR-A in 1979 [21 ] and subsequent cloning in 1990 [1 2 ], signaling by SR-A has been investigated in several studies motivated by demonstration of its role in innate immunity as well as in pathogenesis of atherosclerosis, Alzheimer’s disease, and diabetes-associated pathologies [8 17 22 ]. However, although different effects of SR-A ligands on macrophage signaling functions have been reported [23 24 25 26 27 ], the nonselectivity of the ligands used in most studies precludes certainty as to which particular receptor(s) mediate any of the reported effects. Indeed, many of polyanionic ligands bind to numerous other SRs, within class A (e.g., MARCO) or other subclasses [e.g., LOX-1; refs. 28 29 ]. In some reports, acetylated low density lipoprotein (AcLDL) and malonaldehyde-modified LDL, prototypes of the SR-A-dependent ligands [8 30 31 ], seemed neither to trigger any second messenger activity nor to initiate functional or gene effects upon binding to their preferred receptor on macrophages [15 32 ]. For instance, SR-A-inhibiting concentrations of AcLDL could not induce tumor necrosis factor {alpha} (TNF-{alpha}) production, trigger mobilization of arachidonic acid, or alter LPS stimulation of TNF-{alpha} release [15 ].

In an alternative approach, attempts have been made to deduce SR-A-mediated effects by comparing responses between wild-type and SR-A-deficient macrophages. In general, the results suggest that SR-A-mediated phagocytosis or adhesion is not accompanied by proinflammatory or microbicidal activation of macrophages. For example, although nonopsonic phagocytosis of Neisseria meningitidis was dramatically decreased in bone marrow-derived macrophages from SR-A-deficient mice, TNF-{alpha}, interleukin (IL)-6, IL-10, and IL-12 levels stimulated by these bacteria were not significantly altered [12 ]. Similarly, SR-A was reported primarily responsible for the adhesion of macrophages to oxidized LDL-coated surfaces but not for hydrogen peroxide (H2O2) production under these conditions [33 ]. In contrast, it has been suggested that SR-A-mediated adhesion of rodent microglia and human monocytes to ß-amyloid fibril-coated surfaces stimulates secretion of reactive oxygen species [22 ].

To more precisely dissect SR-A-specific signaling, we have used the anti-SR-A-specific monoclonal antibody (mAb) 2F8 to selectively stimulate mouse macrophages. The data provide a novel demonstration of signaling by SR-A and suggest a potential role for SR-A in modulation of macrophage cytokine responses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies
Low endotoxin/azide-free, rat anti-mouse CD16/32 (Fc{gamma}R II/III clone 2.4G2), anti-CD44 (clone IM7), and irrelevant rat IgG2b (clone A95-1) mAb were obtained from PharMingen (San Diego, CA). Purified (in part of experiments with immobilized mAb) or low endotoxin/azide-free (when used as soluble) rat anti-mouse SR-A 2F8 mAb was purchased from Serotec (Oxford, UK). ImmunoPure goat anti-rat IgG Fc-specific antibody was purchased from Pierce (Rockford, IL) and fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of goat anti-rat IgG (H and L) antibody, from Rockland Immunochemicals (Golbertsville, PA). All chemical reagents not otherwise specified were obtained from Sigma Chemical Co. (St. Louis, MO).

Animals
Female mice, 9–14 weeks old, were used in the experiments. SR-A-deficient mice on the C57BL/6 background [8 ] were maintained in our facility under pathogen-free conditions. C57BL/6 and BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA), C3H/HeJ mice (LPS-nonresponsive strain), from Jackson Laboratories (Bar Harbor, ME), and mice deficient in the {gamma} chain of FcRs on BALB/c background, from Taconic (Germantown, NY).

Cell isolation
For bronchoalveolar lavage (BAL), mice were killed by intraperitoneal (i.p.) injection with 0.25–0.3 ml pentobarbital (Fatal-Plus, Vortech Pharmaceuticals, Dearborn, MI), and BAL was performed with phosphate-buffered saline (PBS; pH 7.4, BioWhittaker, Walkersville, MD) at room temperature. BAL cells were collected into centrifuge tubes kept on ice, and after centrifugation, resuspended in Hanks’ balanced salt solution (HBSS), serum-free medium (Macrophage-SFM; Gibco, Grand Island, NY) or serum-containing medium [RPMI-1640 medium with 25 mM HEPES (Sigma), supplemented with 10% fetal calf serum (FCS; Gemini Bio-Products, Woodland, CA), 2 mM L-glutamine, and a mixture of 0.1 mg/ml streptomycin and 100 U/ml penicillin, FCS-RPMI]. Alveolar macrophages (AMs) constituted at least 99% of BAL cells, as determined by analysis of Giemsa-Wright-stained cytospine preparations. They were cultured at 3 x 105/ml in 0.1 ml HEPES or 0.16–0.2 ml FCS-RPMI.

For peritoneal lavage, mice were quickly killed by inhalation of halothane (Halocarbon Laboratories, River Edge, NJ). Resident peritoneal cells or cells elicited with 1 ml aged, 3% thioglycollate (Difco, Detroit, MI), injected i.p. 5 days earlier, were washed out with PBS and after washing once with PBS, were further processed similarly as BAL cells. Peritoneal cells were cultured at 8–10 x 105/ml.

Receptor cross-linking with immobilized mAb
The antibodies we used against macrophage receptors were generated in the rat and therefore do not bind avidly to protein G. Hence, they were linked to protein G-coated surfaces through a goat antibody, specific for Fc portions of rat IgG. Also, in this system, interaction of Fc regions of IgGs with cellular Fc{gamma}Rs should be at least partially prevented. Goat-anti-rat Fc at 40 µg/ml was immobilized on Reacti-Bind protein G-coated plates (Pierce) by 2.5 h (room temperature) or overnight (4°C) incubation in 100 µl ImmunoPure (G) IgG-binding buffer (PGBB; Pierce). Subsequently, plates were washed two times with 0.2 ml/well PGBB and once with 0.1% bovine serum albumin (BSA; low-endotoxin, IgG-free) in PBS. Rat mAb against mouse macrophage receptors, at 20 µg/ml in 0.1 ml 1% BSA/PBS, were added for 1.5 h incubation at 37°C. Finally, plates were washed four times with HBSS directly before introduction of cell suspensions for H2O2 and cytokine assays described below.

Receptor ligation by soluble mAb
Freshly isolated alveolar or peritoneal cells were incubated in 96-well ultra-low attachment plates (Costar, Corning, NY) with 20 µg/ml mAb in HBSS or FCS-RPMI medium, containing 1 µg/ml LPS (from strain 0127:B8 of Escherichia coli, Sigma) and 20 ng/ml interferon-{gamma} (IFN-{gamma}; murine recombinant, R&D Systems, Minneapolis, MN). Alternatively, thioglycollate-elicited peritoneal cells were suspended in FCS-RPMI medium and plated in 96-well tissue-culture plates. After overnight incubation, nonadherent cells were removed by washing, whereas adherent macrophages were treated as above.

H2O2 assay
H2O2 production was assayed by spectrofluorimetric monitoring of H2O2-mediated horseradish peroxidase (HRP)-catalyzed oxidation of nonfluorescent Amplex Red reagent (Molecular Probes, Eugene, OR) into fluorescent resorufin [34 ]. H2O2 production was assayed in 0.1 ml HBSS containing 3 (AMs) or 10 x 104 (PMs) cells, 50 µM Amplex Red, and 1 U/ml HRP. Fluorescence (535 nm/595 nm) was measured for 40 min every 5 min, starting 2 min after placing a plate in the Spectrafluor Plus (Tecan, Research Triangle Park, NC) spectrofluorometer, prewarmed to 37°C. In initial experiments, amounts of H2O2 released by cells were determined on the basis of standard curves. Since fluorescence intensity was linearly related to H2O2 concentrations exceeding levels produced by cells (data not shown), this step was omitted in subsequent experiments.

For pertussis toxin (PTX) treatment, AMs were suspended in Macrophage-SFM and incubated in 24-well ultra-low attachment polystyrene plates with 1 µg/ml PTX for 4 h at 37°C in a cell culture incubator. Cells were then collected, spun down, resuspended in PTX-containing HBSS, and used in the H2O2 assay.

Release of TNF-{alpha}, IL-6, IL-12 p70, and nitric oxide (NO)
Nitrite, TNF-{alpha}, IL-6, and IL-12 p70 concentrations were determined in supernatants from 20 h cultures of nonadherent cells or tissue-culture plastic-adherent macrophages, stimulated as described above with immobilized or soluble mAb.

Nitrite concentrations in 90 µl aliquots of culture supernatants were determined according to the Griess method. The sensitivity of the method was increased by dissolving sulfonamide in 2 M HCl instead of orthophosphoric acid and by adding individual reagents sequentially [50 µl 1% sulfanilamide, followed by 50 µl 0.1% N-(1-naphthyl)ethylenediamine in deionized water], without premixing. Nitrite concentrations in samples were read from standard curves, generated from serial dilutions of NaNO2. The detection limit of the assay was 0.1–0.2 µM.

IL-6 concentrations in culture supernatants were determined with a sandwich enzyme-linked immunosorbent assay (ELISA) with the use of rat anti-mouse IL-6 mAb MP5-20F3 and biotinylated rat anti-mouse IL-6 mAb MP5-32C11 (PharMingen) as the capture and detecting antibody, respectively. HRP-streptavidin conjugate was obtained from Vector Laboratories (Burlingame, CA), murine IL-6 standard, from PharMingen, and MaxiSorp ELISA plates, from Nunc (Rochester, NY).

IL-12 p70 and TNF-{alpha} determinations were performed with the use of mouse IL-12 p70 or TNF-{alpha} duo set ELISA kits from R&D Systems, according to the manufacturer’s instructions.

Effects of H2O2 donors or scavengers
Macrophages were loaded with glutathione (GSH) by 3 h incubation with 20 mM GSH monoethyl ester (Calbiochem, San Diego, CA) in FCS-RPMI medium. After washing once with HBSS or FCS-RPMI medium, they were used in the H2O2 or cytokine/NO assays, respectively. In other experiments, H2O2 was scavenged by inclusion of 10 kU/ml catalase in the culture medium. Adherent, thioglycollate-elicited peritoneal macrophages (PMs) were preincubated for 1 h with 20 or 50 ng/ml H2O2-generating glucose oxidase, and after washing once, they were stimulated with LPS and IFN-{gamma} in FCS-RPMI medium.

Cell viability
Viability of freshly isolated cells was routinely assessed by their ability to exclude trypan blue. According to this criterion, ≥95% of BAL or thioglycollate-elicited peritoneal cells and essentially all resident peritoneal cells were viable. The effect of treatments on macrophage viability was assessed by measuring lactic dehydrogenase (LDH) release from the cells (Cytotoxicity Detection kit, Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions. In brief, the percent cytotoxicity was calculated as the ratio of LDH in culture supernatants to the total LDH (cell-associated plus that in supernatant) x 100%. Twenty hours treatment with LPS and IFN-{gamma} caused minor cytotoxicity to macrophages, decreasing their viability from 93–90% (~3% cytotoxicity attributable to LPS/IFN-{gamma} treatment). Neither 2F8 nor isotype-matched mAb had any effect on cell viability (data not shown).

Adhesion assay
Divalent cation-independent adhesion of cells to immobilized antibodies was determined at the end of the H2O2 assay. The wells were supplemented with 0.1 ml RPMI-1640 medium, containing 20 mM EDTA (final concentration, 10 mM), and were incubated for a further 25 min at 37°C in a cell-culture incubator. After washing three times with PBS, adherent cells were solubilized with 0.1% Triton X-100 in PBS, and LDH activities in cell lysates were determined with the use of a cytotoxicity detection kit (LDH). Total LDH activity of plated cells was determined in parallel and used to calculate percentage of adherent cells. Pilot studies showed a linear correlation between cell number and LDH values (R2=0.9998) and high sensitivity of the LDH assay (~60 AMs/well for the 30' time of enzymatic reaction per usual protocol).

Flow cytometric analysis of receptor expression
Cells (~5x106) were incubated for 30' with 10 µg/ml receptor-specific or isotype-matched, control mAb in 0.4 ml ice-cold FACS buffer (0.1% NaN3 and 2% FCS in PBS). Subsequently, 2 ml FACS buffer was added, cells were spun down, washed once again, and resuspend in 0.4 ml 50 µg/ml FITC-labeled F(ab')2 of goat anti-rat IgG for 30' incubation on ice. After washing two times, receptor expression was analyzed in the Epics Elite flow cytometer (Coulter, Fullerton, CA).

Statistical analysis
After assessing homogeneity of variances with an F test, means were compared with Student’s t-test for single comparison or ANOVA for multiple comparisons, with the assumption that P values <0.05 indicate statistically significant differences (GraphPad Prism software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Ligation of SR-A or Fc{gamma}RII/III with immobilized mAb stimulates H2O2 production in macrophages
AMs from C3H/HeJ mice, stimulated for 40' with immobilized anti-SR-A 2F8 or anti-Fc{gamma}RII/III 2.4G2 mAb, released, respectively, 1.55 ± 0.08- and 1.98 ± 0.135-fold more H2O2 than cells plated in wells coated with isotype-matched, nonimmune rat IgG2b (Figs. 1A and 2A ). Both 2F8 and 2.4G2 mAb also stimulated H2O2 production significantly in resident peritoneal cells from C3H/HeJ mice (1.66±0.135- and 2.28±0.085-fold, respectively, n=3, data not shown). Enhancement of fluorescence caused by 2F8 mAb-stimulated H2O2 production was first observed after 10–15 min of the assay (Fig. 1) and reached a plateau at ~60 min, likely reflecting cessation of respiratory burst (data not shown). The 2.4G2-stimulated respiratory burst was more rapid (e.g., detected after 5 min) and somewhat larger but reached a plateau earlier, at ~40 min (Fig. 1) .



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  		Figure 1. Effect of anti-SR-A (2F8) and anti-Fc{gamma}RII/III (2.4G2) mAb, immobilized on protein G-coated plates on H2O2 production in AMs from C3H/HeJ (A), C57BL/6 (B), BALB/c (C), and Fc{gamma}RI/III-deficient (D) mice. AMs were plated in mAb-coated plates, and H2O2-mediated, HRP-catalyzed oxidation of Amplex Red reagent was followed spectrofluorimetrically, as described in Materials and Methods. Results of representative single experiments are shown, with fluorescence values produced shown on the y-axis. For comparison, the fluorescence values generated by direct addition of 0.5 µM H2O2 are also plotted (A).

 


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  		Figure 2. (A) Effect of immobilized mAb on H2O2 production by AMs isolated from different strains of mice. Results are presented as means from 4 to 11 (n) independent experiments, each performed in three to four replicates. The summarized data are expressed as percent of H2O2 production (change in fluorescence after 40 min) compared with that seen in wells coated with control IgG2b. *, Significant stimulation over the levels of control IgG2b (P<0.05 in one-sample t-test). (B). EDTA-resistant adhesion of AMs to plates coated with mAb. At the end of the H2O2 assay, EDTA-resistant adhesion to 2F8 or 2.4G2 mAb was determined and expressed as percent of adherent cells over the levels of isotype-matched IgG2b controls (the latter ranging from ~8% of plated cells for Fc{gamma}RI/III–/– AMs to ~35% for SR-A–/– AMs).

 
The effect of 2F8 mAb on H2O2 production resulted specifically from ligation of SR-A because it occurred neither in AMs from SR-A-deficient nor C56BL/6 mice (Figs. 1B and 2A) , the latter strain expressing an allelic isoform of SR-A, which does not bind 2F8 mAb [35 ]. In contrast, in mice deficient with the {gamma} chain of FcRs, which do not express Fc{gamma}RI and III [36 ], 2F8 mAb stimulated H2O2 production (1.303±0.084-fold, Figs. 1D and 2A ) to a similar extent as in their wild-type BALB/c counterparts (1.24±0.045-fold, Figs. 1C and 2A ), whereas the response to 2.4G2 mAb was blunted. Taken together, these results demonstrate that SR-A and Fc{gamma}RIII are linked to stimulation of respiratory burst in murine macrophages.

The specificity of receptor ligation by immobilized mAb was also confirmed in the adhesion assay. Immobilized 2F8 mAb enhanced divalent cation-independent adhesion of C3H/HeJ, BALB/c, and Fc{gamma}RI/III–/– AMs but did not have any effect on C57BL/6 or SR-A–/– AMs (which do not bear epitopes recognized by 2F8). In contrast (and as a further control for these adhesion assays), 2.4G2 mAb supported adhesion of AMs from all strains that express the Fc{gamma}RII/III antigen (C3H/HeJ, BALB/c, SR-A–/–, and C57BL/6) but had no specific effect on adhesion by Fc{gamma}RI/III–/– AMs (Fig. 2B) .

PTX inhibits SR-A- but not Fc{gamma}RIII-stimulated respiratory burst
Involvement of PTX-sensitive heterotrimeric G proteins (Gi/o) in SR-A signaling has been reported [37 38 ]. Pretreatment with PTX (1 µg/ml for 4 h) decreased H2O2 production stimulated by 2F8 mAb and by "nonspecific" adhesion to wells not coated with mAb but not that stimulated by 2.4G2 mAb (Fig. 3A ). In the average from four experiments, PTX decreased SR-A-mediated stimulation of H2O2 production by 75.4 ± 5.86% (P=0.001), whereas that mediated by Fc{gamma}RIII was not significantly affected (Fig. 3B) .



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  		Figure 3. Effect of PTX pretreatment on H2O2 production stimulated in C3H/HeJ macrophages by adhesion to mAb-coated plates. AMs were preincubated with 1 µg/ml PTX for 4 h at 37°C and used in the H2O2 assay described in Materials and Methods. Results of a representative experiment (A) and the average (+SEM) from four experiments, expressed as percentage of IgG2b controls (B), are shown. (B) *, Significant effect of PTX pretreatment (P=0.038).

 
Ligation of SR-A selectively inhibits IL-12 release
The effect of receptor cross-linking with immobilized mAb on IL-6, TNF-{alpha}, and IL-12 p70 release from C3H/HeJ AMs was studied during 20 h incubation in serum-free medium. Cells cultured in wells coated with control IgG2b released similar quantities of IL-6 (21–103 pg/ml) and TNF-{alpha} (0.9–1.4 ng/ml) as those plated in wells coated with 2F8 or 2.4G2 mAb (data not shown). AM adhesion to plates coated only with protein G stimulated IL-12 p70 release (80–180 pg/ml) to an extent similar as that stimulated by LPS + IFN-{gamma} treatment in LPS-normoresponsive AMs (see below), whereas IL-12 release from AMs adhering to uncoated plates was undetectable. Interestingly, the unexpected IL-12 production caused by protein G-only-coated plates was selectively inhibited in the presence of immobilized 2F8 (by 16±2.8%, P=0.03, n=3) but not 2.4G2 or control IgG2b mAb (data not shown).

To determine whether ligation of SR-A inhibits IL-12 release stimulated by the classic activators LPS and IFN-{gamma}, nonadherent AMs were stimulated with LPS/IFN-{gamma} in the presence of soluble 2F8 or control IgG2b mAb. Ligation of SR-A on AMs from BALB/c mice with 2F8 mAb (20 µg/ml) caused 25 ± 4.6% inhibition (P=0.001, n=8) of IFN-{gamma} (20 ng/ml) plus LPS (1 µg/ml)-stimulated IL-12 p70 release (Fig. 4A and B ). In AMs from C57BL/6 mice, 2F8 mAb did not inhibit IL-12 release (Fig. 4B ), demonstrating the SR-A specificity of this response. In contrast to effects on IL-12, neither 2F8 nor isotype-matched, control mAb affected LPS plus IFN-{gamma}-stimulated IL-6 accumulation in the cultures of AMs (Fig. 4C and 4D) . Also LPS plus IFN-{gamma}-stimulated nitrite accumulation in C57BL/6 or BALB/c AM cultures was not affected by any of these mAb (data not shown).



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  		Figure 4. Effect of soluble anti-SR-A 2F8 or control IgG2b mAb on LPS plus IFN-{gamma}-stimulated IL-12 p70 (A, B) and IL-6 (C, D) release in AMs from C57BL/6 mice (C57-AM), AMs from BALB/c mice (Balb-AM), or thioglycollate-elicited PMs from BALB/c mice (Balb-PEM). Nonadherent cells (A–D) or tissue-culture plastic-adherent macrophages (B) were stimulated for 20 h with LPS (1 µg/ml) and IFN-{gamma} (20 ng/ml), with or without indicated mAb (20 µg/ml) in FCS-RPMI medium. IL-6 and IL-12 p70 levels in culture supernatants were determined by ELISA. The data are averages +SEM of n experiments. (B, D) Results are expressed as percentage of cytokine release stimulated by LPS/IFN-{gamma} alone. *, Significant effects of 2F8 mAb (P<0.05).

 
Magnitude of 2F8 mAb effect reflects levels of SR-A expression
We hypothesized that the relatively modest effect of 2F8 mAb, ~25% on both H2O2 (Fig. 2A) and IL-12 (Fig. 4B) release in BALB/c AMs, is linked to a relatively modest level of SR-A expression on normal mouse AMs. This assumption was confirmed by flow cytometric analysis, showing low (as compared with anti-CD44 mAb), uniform labeling of BALB/c AMs with 2F8 mAb [specific mean fluorescence intensity (MFI)=1.6, Fig. 5A ]. To identify a macrophage population with higher SR-A expression, we examined SR-A levels on thioglycollate-elicited PMs. Inflammatory PMs in the fifth day after thioglycollate injection consisted of two distinct, roughly equal-sized populations: Small, monocyte-like cells exhibited very weak expression of SR-A (specific MFI=0.1), whereas expression of SR-A on large macrophages filled with endocytotized material was much higher (specific MFI=9.1) than on AMs (Fig. 5B) . Consistent with our hypothesis, in this mixed population of thioglycollate-elicited peritoneal cells, 2F8 mAb tended to produce stronger inhibition of IL-12 release than in AMs, by 39 ± 11.3% (n=3; Fig. 4B ). The difference between inhibition seen in AMs (n=8) and the inhibition seen in peritoneal cells (n=3) was not statistically significant per se (P=0.1). However, if one outlier out of eight experiments performed on AMs (52% as compared with 11–25% inhibition in the remaining experiments) is excluded, the difference is statistically significant (P=0.013). Even stronger inhibition of IL-12 release was produced by 2F8 mAb in inflammatory PMs, which were purified by overnight adhesion to tissue-culture plastic: 68 ± 4.5% as compared with only 25 ± 1.6% inhibition in similarly treated AMs (P=0.01, n=2, Fig. 4B ).



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  		Figure 5. Flow cytometric analysis of receptor expression on BALB/c AMs (A) or thioglycollate-elicited monocytes–macrophages (B). Cells were labeled with anti-SR-A 2F8 mAb, anti-CD44 IM7 mAb, or isotype-matched, control IgG2b and analyzed by flow cytometry, as described in Materials and Methods. Results of one of two experiments that gave similar results are shown.

 
In resident peritoneal cells from BALB/c mice, treatment with LPS and IFN-{gamma} did not stimulate detectable release of IL-12 p70, and therefore, the effect of 2F8 mAb could not be examined, whereas IL-6 release was not affected by 2F8 or nonimmune IgG2b mAb (data not shown).

H2O2 does not seem to mediate inhibition of IL-12 release caused by SR-A ligation
To determine whether SR-A-stimulated H2O2 is involved in the inhibition of IL-12 release, catalase was included during stimulation of adherent, inflammatory PMs. As H2O2 is a membrane-diffusible molecule, scavenging of extracellular H2O2 by catalase should also decrease its intracellular concentration. Indeed, in rat AMs, exogenous catalase at 200 U/ml almost completely abrogated zymosan-activated serum-stimulated, H2O2-mediated tyrosine phosphorylation and activation of extracellular signal-regulated kinase 1/2 [39 ]. In contrast, exogenous catalase did not affect the 2F8 mAb-mediated inhibition of IL-12 release, even at concentration as high as 10,000 U/ml (Fig. 6A ). The catalase added was validated as biologically active by its inhibition of the increase in H2O2 detected by the Amplex Red assay (as in Fig. 1 , data not shown). Moreover, 2F8 mAb-mediated inhibition of IL-12 release was not mimicked by H2O2 released by 20 ng/ml or 50 ng/ml glucose oxidase (Fig. 6B) . Glucose oxidase at 20 ng/ml released H2O2 with a rate similar to 2F8 mAb-stimulated AMs (data not shown).



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  		Figure 6. Effect of catalase (A) or preincubation with glucose oxidase (B) or GSH (C, D) on anti-SR-A 2F8 mAb-mediated inhibition of LPS/IFN-{gamma}-stimulated IL-12 release (A, B, D) or on stimulation of H2O2 release (C) in thioglycollate-elicited, tissue-culture plastic-adherent BALB/c PMs. Results of single experiments, performed in three to four replicates, representative of at least three similar experiments, are shown. *, Values significantly different from IgG2b controls (P<0.05).

 
Reduced GSH, cooperating with GSH peroxidase and reductase, constitutes the major antioxidant system of macrophages [40 ], which by reducing H2O2-oxidized cysteine residues in proteins, is also involved in the termination of H2O2 signaling [41 ]. In adherent, inflammatory PMs, preincubated for 3 h with a 20-mM membrane-permeable form of GSH, 2F8 mAb-stimulated H2O2 release was completely inhibited (Fig. 6C) . Although consistent with previous reports [42 ], cells preincubated with GSH also released much more IL-12 upon LPS/IFN-{gamma} stimulation, the degree of SR-A-mediated inhibition of IL-12 release was not affected (Fig. 6D) . Taken together, the above results do not support a role for H2O2 in mediating inhibition of IL-12 release upon SR-A ligation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We observed that following ligation with 2F8 mAb, SR-A on mouse macrophages mediates H2O2 production and inhibition of IL-12 release. Responses to 2F8 mAb resulted specifically from ligation of SR-A rather than Fc{gamma}Rs, as evidenced by their lack in C57BL/6 and SR-A-deficient mice and occurrence in Fc{gamma}RI- and III-deficient mice. The lack of 2F8 mAb effects in C57BL/6 and SR-A–/– AMs and conversely, their demonstration in LPS-nonresponsive C3H/HeJ AMs also rule out a significant contribution of contaminating endotoxin. Finally, the magnitude of 2F8 mAb-triggered responses correlated with the level of SR-A expression, as determined by flow cytometric analysis and adhesion of cells to antibody-coated surfaces.

The question of whether SR-A-mediated signaling requires receptor cross-linking has not been addressed directly by the present study. Nevertheless, two observations suggest that extensive cross-linking may not be required. First, we obtained the responses reported using (merely) divalent mAb, and second, AM H2O2 production was stimulated to a similar degree by surface-immobilized (and therefore, possibly causing more cross-linking) 2F8 mAb and soluble antibody (data not shown).

Three types of methodological approaches have been applied to studies of SR-A signaling. These include studying effects of nonselective SR ligands on macrophages [15 24 26 27 32 ] or on cell lines transfected with SR-A [27 38 ]. In the third approach, attempts have been made to deduce SR-A-mediated effects by comparing responses to such ligands between wild-type and SR-A-deficient macrophages [12 33 ]. With one exception [38 ], these methods do not allow drawing definite conclusions about signaling abilities of SR-A. For instance, Peiser et al. [12] found no significant differences in cytokine (including IL-12) responses to N. meningitidis between wild-type and SR-A–/– macrophages. However, SR-A deficiency may be accompanied by compensatory changes in other receptor systems involved in bacteria recognition. Conversely, transfected cells may respond differently than macrophages or be unable to generate macrophage-like responses. Moreover, the ligands may activate endogenous receptors in transfected cells. A good illustration of problems encountered when using transfected cells is the study by Hsu et al. [27 ]. The authors reported stimulation of phosphorylation of unidentified proteins in Bowes human melanoma cells transfected with human SR-A1 by fucoidan and AcLDL. However, the ligands stimulated phosphorylation of the same proteins also in nontransfected cells. Moreover, patterns of protein phosphorylation produced by fucoidan and AcLDL in transfected cells were different from each other and from the pattern produced by these ligands in PMA-differentiated, macrophage-like THP-1 cells.

One major technical obstacle is that nonselective ligands used in studies on SR-A signaling in macrophages may activate a broader range of macrophage SRs [28 29 30 43 44 45 ] as well as receptors not classified into the artificial family of SRs [46 47 ]. Modified LDLs may also be a source of lipid mediators acting through yet another set of receptors [48 ]. Finally, macrophages are known to respond to even trace endotoxin contamination [49 ], whereas such contamination of ligands used to analyze SR-A was present [47 50 ] or not reported [24 26 27 51 ]. Sutterwala et al. [52] reported that mal-BSA-coated erythrocytes inhibited the induction of IL-12 by LPS or LPS + IFN-{gamma} in murine macrophages. The authors reported that SR-A selectivity of mal-BSA-coated erythrocytes had been confirmed by inhibiting their binding to macrophages with receptor-specific mAb or competitive ligands, although neither the data nor details were shown. One concern is that mal-BSA is not a selective ligand of SR-A on macrophages, as it also binds to CD36 and SR-BI, a class B SR [30 ]. At least one of these receptors, CD36, is known to mediate inhibition of IL-12 release [53 ]. Conversely, activation of macrophages by mal-BSA seems mediated by low-affinity receptors, distinct from high-affinity "AcLDL receptor" (SR-A) [32 ]. Similarly, in the reported promotion of Th1-type responses by maleylated antigens, receptors other than SR-A seem to participate, since these responses occurred also when B cells, which do not express SR-A, were used as antigen-presenting cells [54 ].

The use of receptor-specific antibody allowed us to avoid the problems associated with nonspecific, polyanionic SR ligands. However, this approach introduces its own potential specificity issues: effects via FcR ligation and effects from unexpected cross-reactivity with other surface receptors. The former was addressed by use of isotype control IgG, as well as studies in Fc{gamma}R-deficient macrophages. The demonstration of lack of response in SR-A-deficient mice and in a strain, which expresses an allelic form of SR-A that does not react with the 2F8 mAb, allows exclusion of unexpected cross-reactivity.

PTX has been reported to decrease by 46% SR-A-mediated uptake of AcLDL in mouse peritoneal macrophages [37 ] or in human embryonic kidney cells transfected with murine SR-A2 [38 ] and by 37% SR-A-mediated adhesion of transfected cells, with accompanying attenuation of focal adhesion complex formation [38 ]. Our results confirmed involvement of PTX-sensitive Gi/o proteins in signaling by SR-A in mouse AMs. Pretreatment with PTX inhibited by ~75% respiratory burst stimulated in C3H/HeJ AMs by immobilized 2F8 mAb. The mode of SR-A coupling to G protein(s) remains unknown. Extracellular cooperation with some other G protein-coupling receptor(s) is a possibility, as SR-A-mediated cell adhesion and spreading were inhibited by PTX [38 ] but not by deletion of an almost entire cytoplasmatic domain of SR-A [55 ].

H2O2 can act as a second messenger in intracellular signaling. The best-characterized pathways include direct activation of protein kinase C by H2O2, and H2O2-mediated inhibition of specific phosphatases, which enable signaling through protein tyrosine kinases and phosphatidylinositol 3-kinase [56 ]. Activation of some of these pathways is known to inhibit IL-12 production in immune cells [39 42 57 ]. Nevertheless, our results do not support a mechanistic or causal role for H2O2 in the inhibition of IL-12 release in response to SR-A ligation. The inhibition was neither reversed by high concentrations of exogenous catalase nor mimicked by glucose oxidase-derived H2O2. Moreover, loading of macrophages with GSH and completely inhibiting SR-A-stimulated H2O2 release had no effect on SR-A-mediated inhibition of IL-12 release.

The in vivo biological significance of the two macrophage responses identified in our studies remains to be determined. Our data do indicate the feasibility of using mAb assure specificity and to avoid problems inherent in ligation of SRs with potentially polyspecific ligands. Future studies using this antibody-based approach and array analysis approaches may provide additional insights into the signaling and functional consequences of SR-A ligation.


   ACKNOWLEDGEMENTS
 
The study was supported by Grants NIH ES 11008 and 00002.

Received May 4, 2004; revised July 1, 2004; accepted July 13, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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