Published online before print May 29, 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,
,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
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2, 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.
Key Words: apoptosis • phagocytosis • signal transduction • protein kinases/phosphatases • mice • inbred strains
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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), 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 PLC2, the principal PLC isoform in murine Mø, and tyrosine phosphorylation of PLC2 [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 PLC2.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2 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 x 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 x 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 5x 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 x 105 J774 with 2.0 x 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 x 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 1000x 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.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
 View larger version (20K): [in a new window] |
Figure 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 conditions and were transferred to PVDF. Blots were blocked and probed with the anti-SR-A mAb 2F8 diluted in blocker. Blots were then washed for 15 min using Tween-TBS and incubated with HRP-conjugated donkey, anti-rat IgG diluted in blocker, and the signal was detected by ECL. To demonstrate protein loading, blots were then stripped and reprobed using anti-actin antibody. (B) Flow cytometry. J774 were stained with anti-SR-A (filled profile) or with isotype-matched control rat IgG (open profile). Representative histograms are shown. FL2-H, Fluorescence 2-height.
|
 View larger version (29K): [in a new window] |
Figure 2. Anti-SR-A and function-blocking SR-A ligands reduce phagocytosis but not adhesion of AC by the J774 Mø cell line. J774 (1x105 cells/well) were adhered overnight to eight-well chamber slides in medium containing 10% FBS and then were preincubated for 30 min at 37°C and 5% CO2 in medium containing rat IgG (Control), anti-CD36, anti-SR-A (each at 20 µg/ml final concentration), or SR-A ligands (250 µg/ml final concentration). Next, without washing, AC were added as 4 x 106 cells/well for 120 min for phagocytosis assay (A) or as 2 x 107 cells/well for 15 min for adhesion assay (B). Phagocytosis and adhesion were determined by examining H&E-stained slides under oil immersion. (A) Phagocytic index (right panel) and percentage of phagocytic Mø (left panel). Data are mean ± SEM of three to six replicates. *, P < 0.05, compared with control, ANOVA with post-hoc testing by the two-tailed Dunnett test. (B) Adhesion index (right panel) and percentage of adhesion-positive Mø (left panel). Data are mean ± SEM of three replicates per condition in a single experiment.
|
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.
 View larger version (39K): [in a new window] |
Figure 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 extensively, and then lysed in OG buffer. After preclearing, Mø lysates were immunoprecipitated (IP) using anti-Mertk (A and C) or goat anti-SR-A (B) and protein G-agarose, run on a 7.5% acrylamide gel under reducing conditions, and transferred to PVDF. Blots [immunoblots (IB)] were then blocked and probed using rat anti-SR-A (A and C) or anti-Mertk (B) and washed for 15 min using Tween-TBS. After incubation with HRP-conjugated anti-rat or anti-goat IgG in blocker, signal was detected by ECL. Finally, to demonstrate protein loading, blots were stripped and reblotted with anti-Mertk (A and C) or rat anti-SR-A (B). In each case, similar results were obtained in an additional experiment of identical design. wt, Wild-type.
|
2 phosphorylation in J774 in response to AC2 [59
]. Using a molecular approach, the Birge laboratory [65
] has recently demonstrated that Tyr867 of Mertk directly mediates the tyrosine phosphorylation of PLC2 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.
 View larger version (28K): [in a new window] |
Figure 4. Anti-SR-A inhibits the AC-induced tyrosine phosphorylation of Mertk and PLC2. 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 vitro with a fivefold excess of AC for various times as indicated, washed extensively, and then lysed in OG buffer. After preclearing, Mø lysates were (A) immunoprecipitated using anti-pTyr antibody (anti-pY) and protein G-agarose and run on a 7.5% acrylamide gel under reducing conditions and transferred to PVDF or alternatively, (B) run directly on a 7.5% acrylamide gel. Blots were then blocked and probed using anti-Mertk (A) or anti-pMertk (B) and washed for 15 min using Tween-TBS. After incubation with HRP-conjugated anti-goat (for detection of Mertk) or anti-rabbit (for detection of PLC2) IgG in blocker, signal was detected by ECL. Finally, blots were stripped and reblotted with anti-PLC2 (A) or anti-Mertk (B). Similar results were obtained in an additional experiment of identical design.
|
2 phosphorylation in response to AC, by reblotting with anti-PLC2. Some basal phosphorylation of PLC2 was detectable in J774 lysates without exposure to AC, but phosphorylation increased at 5 and 10 min after AC addition (Fig. 4A
, lower gel). PLC2 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 PLC2 [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.
 View larger version (38K): [in a new window] |
Figure 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 to fivefold excess of AC for various times as indicated. Cells were lysed, pTyr immunoprecipitates formed, and gels were probed with anti-Mertk and developed as described in the legend to Figure 3
. The experiments shown were repeated in two additional experiments with similar results. (B) Western blot. PMø lysates (40 µg protein) from wild-type 129P3/J mice or SR-A–/– mice were run on a 4–20% acrylamide gel, and gels were transferred and blocked as described in the legend to Figure 4
and probed with anti-Mertk, followed by stripping and reprobing with anti-actin. (C) Western blot. PMø lysates from wild-type 129P3/J mice or SR-A–/– mice were run on a 4–20% acrylamide gel, and gels were transferred and blocked as described in the legend to Figure 4
and probed with anti-pMertk, followed by stripping and reprobing with anti-Mertk.
|
 View larger version (16K): [in a new window] |
Figure 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 ( ) or SR-A–/– mice ( ) was assayed at various times from 30 to 90 min after addition of a fivefold excess of AC. *, Significantly different; P < 0.05, ANOVA with post-hoc analysis by the two-tailed Dunnett test.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2 [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 PLC2, which by releasing diacylglycerol and inducing Ca++ transients, recruits protein kinase C βII to the nascent AC phagosome [59
, 93
]; and Src-mediated tyrosine phosphorylation of focal adhesion kinase and its recruitment to the 
vβ5 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 PLC2 [65
]. In that study, PLC2 phosphorylation in response to the PS-binding Mertk agonist Gas6 was abrogated completely in the CD8-Mertk Y867F mutant or CD8-MertkKD 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- 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.
Received March 2, 2007; revised March 31, 2008; accepted April 23, 2008.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and attenuates the oxidative burst Cell Death Differ. 13,1533-1540[CrossRef][Medline] receptor function by regulating the expression and function of the common -subunit J. Immunol. 170,4572-4577
}B activation in dendritic cells Blood 109,653-660B Mol. Cell. Biol. 19,1171-11812 during recognition of apoptotic thymocytes by murine macrophages J. Leukoc. Biol. 75,705-713 B transcriptional activation J. Biol. Chem. 283,3618-3627B binding, and transcription of the TNF- gene in murine macrophages J. Leukoc. Biol. 60,784-792[Abstract] and lipopolysaccharide J. Immunol. 172,1825-1832vβ5 integrin-mediated phagocytosis of apoptotic cells J. Cell Sci. 118,539-553 production and lipopolysaccharide-induced endotoxic shock J. Immunol. 162,3498-3503This article has been cited by other articles:
 |
|
 |
|
  A. McColl, S. Bournazos, S. Franz, M. Perretti, B. P. Morgan, C. Haslett, and I. Dransfield Glucocorticoids Induce Protein S-Dependent Phagocytosis of Apoptotic Neutrophils by Human Macrophages J. Immunol., August 1, 2009; 183(3): 2167 - 2175. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
