The scavenger receptor SR-A I/II (CD204) signals via the receptor tyrosine kinase Mertk during apoptotic cell uptake by murine macrophages

doi:10.1189/jlb.0307135

J Leukoc Biol. 2008 August; 84(2): 510–518.

Published online 2008 May 29. doi: 10.1189/jlb.0307135.

PMCID: PMC2718803

The scavenger receptor SR-A I/II (CD204) signals via the receptor tyrosine kinase Mertk during apoptotic cell uptake by murine macrophages

Jill C. Todt,^* Bin Hu,^* and Jeffrey L. Curtis^*^†^‡^§¹

^*Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine,

^†Comprehensive Cancer Center, and

^‡Graduate Program in Immunology, University of Michigan Health Care System, and

^§Pulmonary and Critical Care Medicine Section, Medical Service, Department of Veterans Affairs Care System, Ann Arbor, Michigan, USA

¹ Correspondence: Pulmonary and Critical Care Medicine Section (506/111G), 2215 Fuller Road, Ann Arbor, MI 48105-2303, USA. E-mail: jlcurtis/at/umich.edu

Received March 2, 2007; Revised March 31, 2008; Accepted April 23, 2008.

Abstract

Apoptotic cells (AC) must be cleared by macrophages (Mø) to resolve inflammation effectively. Mertk and scavenger receptor A (SR-A) are two of many receptors involved in AC clearance. As SR-A lacks enzymatic activity or evident intracellular signaling motifs, yet seems to signal in some cell types, we hypothesized that SR-A signals via Mer receptor tyrosine kinase (Mertk), which contains a multisubstrate docking site. We induced apoptosis in murine thymocytes by dexamethasone and used Western blotting and immunoprecipitation to analyze the interaction of Mertk and SR-A in the J774A.1 (J774) murine Mø cell line and in peritoneal Mø of wild-type mice and SR-A−/− mice. Phagocytosis (but not adhesion) of AC by J774 was inhibited by anti-SR-A or function-blocking SR-A ligands. In resting J774, SR-A was associated minimally with unphosphorylated (monomeric) Mertk; exposure to AC induced a time-dependent increase in association of SR-A with Mertk in a direct or indirect manner. Anti-SR-A inhibited AC-induced phosphorylation of Mertk and of phospholipase Cγ2, essential steps in AC ingestion. Relative to tissue Mø of wild-type mice, AC-induced Mertk phosphorylation was reduced and delayed in tissue Mø of SR-A−/− mice, as was in vitro AC ingestion at early time-points. Thus, during AC uptake by murine Mø, SR-A is essential for optimal phosphorylation of Mertk and subsequent signaling required for AC ingestion. These data support the Mertk/SR-A complex as a potential target to manipulate AC clearance and hence, resolution of inflammation and infections.

Keywords: apoptosis, phagocytosis, signal transduction, protein kinases/phosphatases, mice, inbred strains

INTRODUCTION

Efficient clearance of apoptotic leukocytes in resolving inflammation is vital to minimize tissue injury and to avoid the risk of autoimmunity as a result of inappropriate presentation of self-antigens [1,2,3]. In some organs, apoptotic cells (AC) can clearly be ingested by cell types other than macrophages (Mø), especially certain specialized epithelial cells [4,5,6,7]. In the lungs, a frequent site of infection and inflammatory diseases, epithelial cells and fibroblasts ingest AC in vitro, albeit less efficiently than do Mø [8,9,10]. However, during steady-state in most organs and certainly during resolving inflammation, mononuclear phagocytes are believed to clear most AC. Importantly, Mø that contact AC assume a unique activation state [11,12,13,14,15,16,17,18] and elaborate TGF-β and PGE₂ [13, 19]. These immunosuppressive mediators hasten the resolution of inflammation from successfully managed infections [20] but also have the potential to compromise host defenses against subsequent pathogens and to promote fibrosis [21,22,23,24,25]. Thus, understanding the molecular mechanisms mediating Mø ingestion of AC has significant implications for multiple areas of biology and medicine.

No current model comprehensively explains Mø uptake of AC, a process initiated by changes on the AC surface (referred to as “eat me” signals) that induce its adhesion to the phagocyte. This interaction is mediated by phagocyte receptors that bind the AC directly or via a variety of opsonizing proteins, which bridge apoptotic ligands and the receptor. The best-characterized AC eat-me signal is the exposure of phosphatidylserine (PS) on the outer leaflet of the AC plasma membrane [26]. The “tether and tickle” model of AC ingestion [27] postulates that initial AC adhesion via multiple redundant receptors is followed by triggering via stereospecific recognition of the exposed PS [28]. Although it has since become evident that the original gene identified as this PS receptor [29] appears not to encode a cell-surface receptor (reviewed in ref. [30]), three groups have recently identified novel and unrelated PS-binding receptors: T cell Ig- and mucin domain-containing molecule [31], brain-specific angiogenesis inhibitor 1 [32], and stabilin-2 [33]. It appears likely that PS recognition will prove to be central to AC ingestion [34].

However, a bewildering array of other phagocyte receptors of unrelated molecular classes has also been implicated in AC uptake [26, 35, 36], in some cases based on antibody-blocking not corroborated by more definitive experiments using gene-targeting or peptide-domain modification. Such putative AC receptors include lectins, the vitronectin receptor, scavenger receptors of several classes, CD14, and CD91 (reviewed in ref. [37]). The key question of how these many receptors may interact to induce AC ingestion and subsequent immunoregulatory effects is only beginning to be understood [15, 38, 39]. That some of these receptors may be nonessential for the decision by the phagocyte to ingest the AC is suggested by the finding that expression of individual receptors can increase AC adhesion with minimal effects on uptake [40]. One reason for this apparent redundancy might be that receptor expression and possibly use are tissue-, cell type-, and condition-dependent [41]. An alternative, not mutually exclusive, reason could be that efficient Mø ingestion of AC benefits from the integration of signals from multiple receptors, some possibly not specific for the apoptotic state.

Scavenger receptor A (SR-A) I/II is a multifunctional Mø receptor [42, 43] that is involved in AC clearance, although not obligatorily [44,45,46]. SR-A, also known as CD204, is a helically coiled, homotrimeric, type II integral membrane protein, which does not bind unmodified PS [47]. SR-A does bind a wide variety of ligands, most of which are covalently modified molecules [e.g., oxidized low-density lipoprotein (LDL)] distinguished from their native counterparts in part by electrostatic charge [48]. Such modifications include oxidation and glycation that may be particularly prevalent under conditions such as cellular stress and death. Importantly, the amino acid sequence of SR-A contains no evident motifs associated with intracellular signaling. Thus, despite evidence that SR-A ligation not only induces AC ingestion but also regulates cytokine and chemokine elaboration [49,50,51], how SR-A signals and whether it is yet another potentially redundant AC adhesion receptor are unknown.

By contrast, Mer receptor tyrosine kinase (Mertk; also known as Tyro12, c-mer proto-oncogene tyrosine kinase, Eyk, and NYK; Swissprot accession number Q12866) is essential for AC uptake by murine Mø in vivo and in vitro [3, 52, 53]. Mertk is a large (200-kDa) transmembrane receptor that binds PS indirectly [via the serum-derived protein growth arrest-specific gene 6 (Gas6)] and possesses well-described signaling capabilities [54]. Mertk contains extracellular Ig and fibronectin III domains (implying possible cell–cell interactions) and an intracellular, multisubstrate docking site that can bind Src homology 2-containing proteins such as the p85 chain of the enzyme PI-3K and the adapters Grb2 and Vav1 [55, 56]. As a receptor tyrosine kinase (RTK) of the Tyro3 family, Mertk can phosphorylate and activate multiple downstream signaling intermediaries. Stimulation of a chimeric receptor containing the Mertk intracellular domain phosphorylated PI-3K p85, Grb2, Raf-1, Shc, phospholipase C (PLC)γ, and ERK [57]. We have previously reported that Mertk is not required for AC adhesion by murine tissue Mø and that exposure of murine peritoneal Mø (PMø) to AC sequentially induces tyrosine phosphorylation of Mertk, association of Mertk with PLCγ2, the principal PLCγ isoform in murine Mø, and tyrosine phosphorylation of PLCγ2 [58, 59]. Collectively, these characteristics indicate that Mertk plays a central role in signaling during AC recognition.

We hypothesized that on Mø contact with AC, SR-A might interact with and signal via Mertk. To test this hypothesis, we examined the interactions of SR-A and Mertk in murine Mø cell line J774A.1 (J774), which avidly ingests AC in a Mertk-dependent manner [59, 60], and in resident murine tissue Mø of wild-type and SR-A gene-targeted mice. Using immunoprecipitation, Western blotting, and functional assays of in vitro phagocytosis and adhesion, we provide evidence that SR-A signaling during AC ingestion involves Mertk and PLCγ2.

MATERIALS AND METHODS

Reagents

The following reagents were purchased from the indicated vendors: PBS without cations, DMEM, FBS, and penicillin/streptomycin from Life Technologies (Rockville, MD, USA); sodium orthovandadate, sodium fluoride, octyl-β-D-glucopyranoside (OG), fucoidan, heparin, dextran sulfate, BSA Fraction V, dexamethasone, 2-ME, glycerol, NaCl, Tris HCl, and phosphatase inhibitor cocktail II from Sigma Chemical Co. (St. Louis, MO, USA); rat anti-SR-A antibody (clone 2F8) from Serotec (Raleigh, SC, USA); goat anti-SR-A antibody and polyclonal goat anti-Mertk antibody from R&D Systems (Minneapolis, MN, USA); anti-CD36 from BD PharMingen (San Jose, CA, USA); polyclonal rabbit anti-PLCγ2 antibody (sc-407), anti-actin antibody, rat IgG, mouse IgG, protein L-agarose, protein G-agarose, and HRP-conjugated anti-rabbit and anti-goat IgG from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-phospho (p)Mertk antibody from FabGennix Inc. (Frisco, TX, USA); SDS, 0.2 μm polyvinylidene difluoride (PVDF) membrane, nonfat dry milk blocker, and 7.5% acrylamide Ready Gels from BioRad (Hercules, CA, USA); Supersignal West Femto maximum sensitivity substrate and TBS, pH 7.2, from Pierce (Rockford, IL, USA); and Kodak X-Omat AR film and eight-well Lab-Tek slides from Fisher Scientific (Chicago, IL, USA).

Mice

SR-A−/− mice on a 129/SvJ strain background, the generous gift of Dr. Willem J. S. de Villiers (University of Kentucky Medical Center, Lexington, KY, USA), were bred locally at our facility. Specific pathogen-free, female mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA; 129P3/J strain) or Charles River Laboratories Inc. (Wilmington, MA, USA; C57BL/6 strain) at 7–8 weeks of age and were used at 8–14 weeks of age. Mice were housed in the Animal Care Facility at the Ann Arbor VA Medical Center (Ann Arbor, MI, USA), which is fully accredited by the American Association for Accreditation of Laboratory Animal Care. This study complied with the National Institutes of Health “Guide for the Care and Use of Laboratory Animals” (Department of Health, Education, and Welfare, Publication No. 80-32) and followed a protocol approved by the Animal Care Subcommittee of the local Institutional Review Board.

Preparation of AC

All thymocytes were obtained from unmanipulated C57BL/6 mice, which were killed humanely by rapid asphyxia in a high CO₂ environment. Thymuses were harvested and minced to yield a single-cell suspension. To induce apoptosis, thymocytes were resuspended in complete medium to a concentration of 1 × 10⁶ cells/ml and incubated for 5 h in complete medium containing 1 μM dexamethasone. This treatment yields a population with a low degree of contamination by late apoptotic or necrotic cells [60, 61].

Preparation of activated Mø and PMø

129/SvJ SR-A−/− mice and wild-type 129P3/J mice were killed by asphyxia in a high CO₂ environment. PMø were collected by peritoneal lavage using a total of 10 ml PBS-EDTA, which was administered in 2 ml aliquots. PMø among the lavage cells were isolated by plastic adherence with an overnight incubation in complete medium in a 5% CO₂ environment at 37°C. Nonadherent cells were removed by gentle washing.

Cell line

The J774 cell line was obtained from American Type Culture Collection (Manassas, VA, USA). These Mø were grown in 150 cm² plastic flasks in DMEM high-glucose medium containing 10% heat-inactivated FCS and 100 units/ml penicillin/streptomycin. The cells were incubated at 37°C in air (95%)-CO₂ (5%) in a humidified incubator. Cells were removed from the flasks by scraping and plated at 10–15 × 10⁶ in a 100-mm plate (for immunoprecipitation and Western blot) and used for experiments (immunoprecipitation, Western blot, phagocytosis, and adhesion assays) after 24 h adherence.

Immunoprecipitation

For immunoprecipitation studies, J774 cells or Mø from SRA−/− mice or wild-type 129P3/J mice were exposed to apoptotic thymocytes in a 1:5 ratio for various times. For blocking experiments, J774 were preincubated with anti-SR-A or rat IgG (20 μg/ml) for 45 min. For lysis, J774 were washed gently three times using cold PBS with 10 mM sodium vanadate and 50 mM sodium fluoride (phosphatase inhibitors) and then lysed for 30 min on ice using cold OG buffer, which contained 1.5% OG, 50 mM Tris HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 10% glycerol, protease inhibitor cocktail II (1:100, Sigma Chemical Co.), and phosphatase inhibitors, as above. Cells were drawn twice into a 3-ml syringe via a 25-G needle. The lysed cells were centrifuged at 13,000 g for 10 min at 4°C. To preclear lysed cell supernatants, one of the following combinations was added: 5 μg mouse IgG and 40 μl protein G agarose (for phosphorylation studies) or 5 μg goat IgG and 40 μl protein G-agarose (for association studies). Cell lysates were rocked for 30 min at 4°C and then were centrifuged at 2090 g for 5 min at 4°C. The pellets were washed twice in OG buffer, and the supernatants were combined. The immunoprecipitating antibody (6 μg) was added to the combined supernatants, and the mixture was rocked overnight at 4°C. Next, 40 μl protein G agarose was added, and the mixture was rocked for 2 h at 4°C. Finally, the protein bound to the agarose conjugate was centrifuged at 2090 g for 5 min at 4°C, and the pellet was washed twice using OG buffer.

Western analysis

To run samples on SDS-PAGE, 15 μl 5× SDS-PAGE sample buffer and 7 μl 1 M DTT were added to the pellet from immunoprecipitation, and samples were heated at 90°C for 5 min. The samples were centrifuged at 2090 g for 5 min at room temperature, and the supernatant was saved for SDS-PAGE. Protein from an equal number of cells (for immunoprecipitation and expression of pMertk) or an equal amount of protein (for expression of SR-A or Mertk) was loaded onto 7.5% acrylamide Ready Gels (Bio-Rad), run at 150 V, and transferred to 0.2 μm PVDF sequencing grade overnight at 30 V in 20% methanol, 25 mM Tris HCl, and 192 mM glycine. Blots were blocked in 5% milk, 0.1% Tween TBS (blocker) for 45 min at room temperature. Primary antibody was added in optimal dilution in blocker and incubated overnight at 4°C. Blots were washed five times for 15 min each using Tween-TBS. Secondary antibody was added in blocker, incubated for 45 min at room temperature, and washed five times for 15 min each using TBS-Tween. Blots were stained for 5 min at room temperature using Pierce Supersignal West Femto detection systems. Control blots stained with the secondary antibody alone showed no staining.

Flow cytometry

J774 were washed twice in staining buffer (Difco, Detroit, MI, USA), resuspended in 100 μl staining buffer, and incubated for 30 min at 4°C in the dark with labeled antibodies diluted in 100 μl staining buffer. Final antibody concentrations were 1–2 μg/10⁶ cells. FcR was blocked using mAb 2.4G2 (anti-CD16/32) for all primary mAb. After incubation, cells were washed twice in staining buffer, resuspended in 0.5 ml staining buffer, and analyzed immediately. Flow cytometry was performed as described previously in detail [62] using a FACScan cytometer (Becton Dickinson, Mountain View, CA, USA) running CellQuest software on a PowerPC microcomputer (Apple, Cupertino, CA, USA) for data collection and analysis. A minimum of 10,000 viable cells was analyzed to determine cell-surface receptor expression.

Phagocytosis assay

AC phagocytosis in vitro was assayed by coincubation of 1.0 × 10⁵ J774 with 2.0 × 10⁶ apoptotic thymocytes for 120 min (J774) at 37°C in 5% CO₂ as described previously [60]. Results are expressed as percentage of J774 containing at least one ingested AC (percent phagocytosis) and as phagocytic index, which was generated by multiplying the percentage of phagocytosis by the mean number of ingested AC per Mø. Antibody (20 μg/ml) or SR-A ligands (150 μg/ml) were added 30 min before addition of AC at concentrations previously found to be inhibitory.

Adhesion assay

Adherence of AC to J774 in vitro was assayed in the same manner as phagocytosis, except that 1 × 10⁷ apoptotic thymocytes were added to each well, yielding a 100:1 thymocyte:Mø ratio [58]. The slides were incubated for 15 min at 37°C and then washed in a standardized manner by dipping individual slides in each of two Wheaton jars filled with ice-cold PBS, and stained using H&E (Richard-Allan, Kalamazoo, MI, USA). These assay conditions have been found to be optimal (unpublished observation). In previously published experiments, we definitively excluded a contribution of ingested cells at this time-point using a fluorescence approach that permitted complete quenching of extracellular AC [58]. Additionally, we verified that under these conditions, interference by any viable thymocytes in the AC mixture is negligible [58]. Adhesion was evaluated by counting 200 J774 per well at 1000× magnification under oil immersion and scoring for bound thymocytes. Results are expressed as percentage of J774 binding at least one AC (percent adhesive Mø) and as adhesion index, which was generated by multiplying the percentage of adherence-positive Mø by the mean number of adherent AC per Mø.

Statistical analysis

Data are expressed as mean ± sem. Statistical calculations were performed using Statview 5.0 (SAS Institute, Cary, NC, USA) on a Macintosh Power PC G4 computer. Continuous ratio scale data were evaluated by ANOVA with post-hoc analysis by the two-tailed Dunnett test, which specifically compares treatment groups with a control group [63]. Use of this parametric statistic was deemed appropriate, as phagocytosis of apoptotic thymocytes by PMø has been shown to follow a Gaussian distribution [64]. Significant differences were defined as P < 0.05.

RESULTS

SR-A is expressed strongly on the surface of J774 Mø but not by apoptotic thymocytes

To determine the relative expression of SR-A by J774 and apoptotic thymocytes, we used Western blot analysis of equal amounts of protein from each cell type (Fig. 1A

). Results confirmed that J774 strongly expressed SR-A. Apoptotic thymocytes did not express SR-A (Fig. 1A)

, making it unlikely that any residual, adherent thymocytes remaining after washing would contribute to our analysis of SR-A. Flow cytometric analysis confirmed that J774 expressed SR-A on the cell surface (Fig. 1B)

Fig. 1.

SR-A is expressed strongly by J774 but not by AC. (A) Western blotting. The murine Mø cell line J774 and apoptotic thymocytes (AC) from normal C57BL/6 mice were lysed, and equal amounts of protein were run on a 7.5% polyacrylamide gel under reducing (more ...)

Anti-SR-A and SR-A ligands significantly reduce J774 phagocytosis but not adhesion of AC

To verify the importance of SR-A in the phagocytosis of AC by the J774 cell line, we measured phagocytosis in the presence of anti-SR-A, control rat IgG, or functional SR-A ligands. Pretreatment with anti-SR-A or with the SR-A ligands dextran sulfate and fucoidan significantly inhibited phagocytosis of AC by J774, whereas pretreatment with the control compounds BSA, heparin, and anti-CD36 did not (Fig. 2A

). By contrast, SR-A inhibition had no effect on adhesion of AC by J774 (Fig. 2B)

. Thus, J774 appears to use other receptors for AC adhesion but uses SR-A in signaling, which leads to AC uptake in agreement with previous reports using thymic Mø in vitro [44].

Fig. 2.

Anti-SR-A and function-blocking SR-A ligands reduce phagocytosis but not adhesion of AC by the J774 Mø cell line. J774 (1×10⁵ cells/well) were adhered overnight to eight-well chamber slides in medium containing 10% FBS and then were preincubated (more ...)

SR-A and Mertk physically associate in response to AC

To determine whether SR-A and Mertk associate in response to exposure to AC, we used an immunoprecipitation approach (Fig. 3

). We found that there was slight association of SR-A and Mertk in untreated J774 but that association increased after AC addition at 5, 10, and 30 min and regressed at 60 min (Fig. 3A)

. This finding was confirmed by reversing the IP and IB antibodies, which showed slight association of SR-A and Mertk in untreated J774 but increased association at 5 and 10 min after AC addition (Fig. 3B)

. Exposure to AC also induced increased association of Mertk and SR-A in PMø from wild-type mice (Fig. 3C)

; as expected, no band was detected regardless of exposure to AC in PMø of SR-A−/− mice, which verified that the band seen at ~89 kDa was indeed SR-A. There was no association of Mertk and SR-A in preparations of apoptotic thymocyte alone (data not shown), as that cell type lacks Mertk and SR-A. Therefore, exposure to AC induces the association of SR-A and Mertk (which could be direct or via another intermediary protein), potentially focusing the kinase on specific intracellular substrates.

Fig. 3.

Exposure of J774 to AC induces association of SR-A and Mertk. Equal numbers of J774 (A and B) or PMø from 129P3/J or SR-A−/− mice (C) were stimulated in vitro with a fivefold excess of AC for various times as indicated, washed (more ...)

SR-A inhibition blocks Mertk and PLCγ2 phosphorylation in J774 in response to AC

We previously reported that after AC, adhesion to Mø Mertk becomes tyrosine-phosphorylated (an event essential for development of its kinase activity as well as to activate its multisubstrate-docking site), followed by physical association with and tyrosine phosphorylation of PLCγ2 [59]. Using a molecular approach, the Birge laboratory [65] has recently demonstrated that Tyr867 of Mertk directly mediates the tyrosine phosphorylation of PLCγ2 in response to the AC-binding Mertk ligand Gas6. Therefore, having shown that SR-A and Mertk associate, we asked whether anti-SR-A could block these sequential phosphorylation events. Immunoprecipitation with anti-pTyr and Western blotting with anti-Mertk showed no detectable pMertk at baseline, but rapid induction of phosphorylation in J774 exposed to apoptotic thymocytes for 5, 10, and 20 min (Fig. 4A

, upper gel). This AC-induced phosphorylation of Mertk was substantially inhibited by soluble anti-SR-A at 5 and 10 min and was strongly reduced at 20 min. To confirm these results, we performed Western blotting with anti-pMertk of J774 cell lysates exposed to AC. These lysates showed limited pMertk at baseline but induction of phosphorylation at 5, 10, and 20 min (Fig. 4B

, upper gel). Once again, AC-induced phosphorylation of Mertk was substantially inhibited by soluble anti-SR-A at 5 and 10 min and at 20 min. The rat IgG did seem to induce some Mertk phosphorylation in comparison with control conditions lacking any Ig, presumably as a result of Fc effects. Collectively, these results imply that SR-A function is essential for the prompt and optimal activation of Mertk by AC.

Fig. 4.

Anti-SR-A inhibits the AC-induced tyrosine phosphorylation of Mertk and PLCγ2. Equal numbers of J774 were preincubated at 37°C and 5% CO₂ in medium containing 20 μg/ml rat IgG or anti-SR-A and after washing, were stimulated in (more ...)

We next examined whether anti-SR-A also blocks PLCγ2 phosphorylation in response to AC, by reblotting with anti-PLCγ2. Some basal phosphorylation of PLCγ2 was detectable in J774 lysates without exposure to AC, but phosphorylation increased at 5 and 10 min after AC addition (Fig. 4A

, lower gel). PLCγ2 phosphorylation in response to AC was almost entirely blocked by anti-SR-A preincubation. We have previously reported that apoptotic thymocytes themselves show undetectable phosphorylation of PLCγ2 [59].

Mertk phosphorylation in response to AC exposure is reduced in Mø of SR-A−/− mice

It was previously reported that SR-A−/− Mø show a 50% reduction in apoptotic thymocyte uptake in vitro [44]. To correlate this finding with changes in SR-A signaling through Mertk, we exposed PMø from wild-type mice or SR-A−/− mice to AC and measured Mertk tyrosine phosphorylation, which increased in PMø of wild-type mice at 10, 30, and 60 min after AC exposure, whereas this increase was reduced severely in PMø of SR-A−/− mice (Fig. 5A

). Western blotting showed that this difference was not a result of inadequate expression of Mertk in SR-A−/− mice, in which the RTK was actually relatively increased (Fig. 5B)

. To confirm these results using an alternative design, we also exposed PMø of SRA−/− mice and wild-type mice to AC in vitro and performed Western blotting with anti-pMertk. Wild-type PMø lysates showed limited pMertk at baseline but induction of phosphorylation at 10 and 20 min. This AC-induced phosphorylation of Mertk was inhibited substantially in SR-A−/− PMø (Fig. 5C)

. Therefore, optimal tyrosine phosphorylation of Mertk in response to AC in vitro in mature murine PMø is dependent on SR-A expression.

Fig. 5.

Mertk phosphorylation is inhibited in tissue Mø of SR-A−/− mice. (A) Coimmunoprecipitation of PMø following AC exposure. PMø from 129P3/J mice (upper gel) or SR-A−/− mice (lower gel) were exposed (more ...)

Ingestion of AC by PMø of SR-A−/− mice is delayed but not abolished

Finally, we examined how genetic absence of SR-A influenced the kinetics of AC ingestion in vitro, using resident PMø from untreated mice. Results showed that the phagocytic index was reduced significantly at early time-points (30 and 45 min) in PMø of SR-A−/− mice but that there was no significant difference at 60 min (Fig. 6

). The eventual ability of Mø to ingest AC, despite the absence of SR-A in this kinetic experiment, correlates with the absence of an AC clearance defect in vivo [45], an example of the redundancy in AC clearance mechanisms.

Fig. 6.

AC phagocytosis by resident PMø is delayed in the absence of SR-A. In vitro phagocytosis of AC by resident PMø from wild-type mice ( [filled square]

) or SR-A−/− mice (○) was assayed at various times from 30 to 90 min after (more ...)

DISCUSSION

The results of this study indicate that during AC-induced signaling and the resulting phagocytosis of AC by murine Mø, SR-A rapidly, physically associates with the receptor Mertk, directly or indirectly, and is essential for optimal Mertk activation. Soluble anti-SR-A blocked two known key early events during AC ingestion: the sequential tyrosine phosphorylation of Mertk and of PLCγ2 [59] as well as AC phagocytosis itself. AC-induced tyrosine phosphorylation of Mertk on tissue Mø of SR-A−/− mice was reduced in magnitude and duration relative to Mø of wild-type mice. We also found that SR-A, like Mertk itself, was not essential for Mø adhesion of AC, which is noteworthy given the known role of SR-A in mediating cation-independent adhesion to a wide range of substrates [66,67,68]. These results advance understanding of the earliest signaling steps in phagocytosis of AC by Mø, an event with important immunoregulatory consequences.

Defining how SR-A participates in Mø signal transduction is particularly important as a result of the central role of this highly versatile scavenger receptor in host defense, atherogenesis, and neoplasia [68,69,70,71,72]. The most common approach to this question has been to examine effects of nonselective SR ligands, such as fucoidan, polyinosinic:polycytidylic acid, or acetylated LDL using Mø [73,74,75,76] or cell lines transfected with SR-A [75, 77]. Interpretation of these studies is hampered by the lack of specificity of such ligands, which can bind to other scavenger receptors. This caveat remains even when they are tested on SR-A−/− Mø [78, 79] as a result of compensatory up-regulation of alternative receptors [46]. To avoid these issues, we, like other recent investigators [49], primarily used the specific function-blocking mAb 2F8. As this approach has potential problems as a result of FcR ligation, we used IgG as a control in the blocking experiments.

Our finding of a role for SR-A in Mø uptake of AC in vitro agrees with and extends previous results using mAb 2F8 on thymic and elicited Mø of wild-type mice and Mø from SR-A−/− mice [44]. The exact role of SR-A in AC clearance in vivo remains uncertain. SR-A−/− mice have no developmental abnormalities [46] and show normal clearance of AC within the thymus [45] (although clearance in other organs has not been reported). However, these observations might reflect compensatory changes in alternative AC receptors, such as CD36, which is up-regulated in fetal Mø of SR-A−/− mice [80], or efficient AC uptake by cell types other than Mø [81], as occurs in some organs [5, 7, 8, 82]. It is clear that SR-A is strongly expressed primarily by cell types that avidly engulf AC [46, 83]. These results are, to our knowledge, the first demonstration of an interaction (direct or indirect) between SR-A and a RTK. A variety of previous studies [75, 76, 84, 85] has provided indirect evidence that SR-A associates with tyrosine kinases, based in most cases on inhibitor studies and use of nonspecific SR-A ligands. Although we did find that one related RTK, STK/RON [86], was not activated on direct activation of SR-A using immobilized anti-SR-A (data not shown), it is unlikely that Mertk is unique in associating with SR-A; e.g., the cytoplasmic tyrosine kinase Lyn was coimmunoprecipitated using polyclonal rabbit anti-human SR-A antiserum within 30 s of exposing the Mø cell line THP-1 to acetyl LDL [87]. Conversely, SR-A is a target of serine phosphorylation by JNK2 [88], and Gas6, a Mertk ligand, can induce SR-A expression in human smooth muscle cells via a pathway requiring the serine/threonine kinase AKT [89].

Our findings imply that SR-A participates in optimal AC uptake by aggregating Mertk monomers to facilitate their autotransphosphorylation. Such an accessory action of SR-A could be important to initiate Mø signaling, which leads to phagocytosis of AC, as the Biacore assay has shown that Mertk interacts with low avidity with its soluble AC-binding intermediaries, Gas6 and protein S [90,91,92]. Activated Mertk provides multiple signals to initiate AC uptake, including release of VAV1 constitutively bound to inactive Mertk, freeing that guanine exchange factor to hydrolyze the RhoA family members [56]; activation of PLCγ2, which by releasing diacylglycerol and inducing Ca⁺⁺ transients, recruits protein kinase C βΙΙ to the nascent AC phagosome [59, 93]; and Src-mediated tyrosine phosphorylation of focal adhesion kinase and its recruitment to the αvβ5 integrin [94]. This final step is particularly important, as it induces formation of the p130(CAS)/CrkII/Dock 180 signaling complex that activates Rac-1 and hence, actin polymerization. A recent paper identified Tyr867 of Mertk as required for activation of PI-3K and PLCγ2 [65]. In that study, PLCγ2 phosphorylation in response to the PS-binding Mertk agonist Gas6 was abrogated completely in the CD8-Mertk Y867F mutant or CD8-Mertk^KD cells. The many consequences of Mertk activation place it at the center of the AC uptake process. Hence, data from this study and others, showing that SR-A is not obligatory for efficient AC uptake [44,45,46], imply that other receptors will be able to increase the efficiency of Mertk autotransphosphorylation during AC recognition.

A final aspect of these results is the insight they provide on the unique activation state induced by Mø contact with AC [11,12,13,14,15,16,17,18,19,20]. SR-A was the first pattern recognition receptor to be shown to counteract proinflammatory receptors [50, 95, 96], although the signaling pathways involved remain incompletely defined. SR-A−/− mice and gene-targeted mice with defective Mertk expression exhibit increased sensitivity to endotoxin-induced shock, related to increased TNF-α production [95, 97]. Significantly, two groups have recently shown an essential role for Mertk, along with other members of the Tyro3 family of RTKs, to which it belongs, for the ability of AC exposure to block subsequent proinflammatory cytokine production by dendritic cells (DC) [39, 98]. Whether immunosuppressive effects of SR-A activation by others of its many ligands depend on signaling via Mertk will require further study.

In summary, we provide evidence that SR-A, a receptor with broad ligand-binding properties and a central role in host defense, pairs with Mertk during signaling induced by AC, which leads to Mø ingestion of AC. This intermolecular interaction, likely induced when the two receptors bind their own cognate ligands on the AC surface, illustrates how a receptor with multiple ligands can participate in delivery of a specific signal through its binding partners. Defining the exact importance of SR-A in uptake of AC by Mø and DC in different organs will require additional in vivo experimentation. Understanding the signaling pathways induced during AC clearance is a first step to manipulate the process therapeutically.

Acknowledgments

RO1 HL056309 and RO1 HL082480 from the U.S. Public Health Service and Merit Review funding and a Research Enhancement Award Program (REAP) grant from the Department of Veterans Affairs supported this work. We thank all of the members of the Ann Arbor VA REAP for helpful suggestions and discussions; Dr. Willem J. S. de Villiers (University of Kentucky Medical Center, Lexington, KY, USA) for the generous gift of SR-A−/− mice; and Joyce O’Brien for secretarial support.

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