Serum Amyloid P Component Binds to Fc Receptors and Opsonizes ...

Serum Amyloid P Component Binds to Fc␥ Receptors and Opsonizes Particles for Phagocytosis1 Dwaipayan Bharadwaj,*† Carolyn Mold,‡ Eric Markham,‡ and Terry W. Du Clos2*† Serum amyloid P component (SAP) is a member of the pentraxin family of proteins. These proteins are characterized by cyclic pentameric structure, calcium-dependent ligand binding, and frequent regulation as acute-phase serum proteins. SAP is the serum precursor of the P component of amyloid. It binds to a broad group of molecules, including autoantigens, through a pattern recognition binding site. The related pentraxin, C-reactive protein (CRP), is a strong acute-phase reactant in man and an opsonin. We previously determined that the binding of CRP to leukocytes occurs through Fc receptors for IgG (Fc␥R). We now report that SAP also binds to Fc␥R and opsonizes particles for phagocytosis by human polymorphonuclear leukocytes (PMN). Specific, saturable binding of SAP to Fc␥RI, Fc␥RIIa, and Fc␥RIIIb expressed on transfected COS cells was detected using SAP-biotin and PE-streptavidin. Zymosan was used to test the functional consequences of SAP and CRP binding to Fc␥R. Both SAP and CRP bound to zymosan and enhanced its uptake by PMN. This enhanced phagocytosis was abrogated by treatment of PMN with wortmannin, a phosphatidylinositol-3 kinase inhibitor, or with piceatannol, a Syk inhibitor, consistent with uptake through Fc␥R. Treatment of PMN with phosphatidylinositol-specific phospholipase C to remove Fc␥RIIIb also decreased phagocytosis of SAPopsonized zymosan, but not CRP-opsonized zymosan. These results suggest that SAP may function in host defense. In addition, as SAP binds to chromatin, a major immunogen in systemic lupus erythematosus, it may provide a clearance mechanism for this Ag through Fc␥R bearing cells. The Journal of Immunology, 2001, 166: 6735– 6741.

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he innate immune system is responsible for the recognition of bacteria, fungi, and a variety of other pathogens. In addition, the innate immune system may be responsible for the recognition of necrotic and apoptotic cells and self Ags that are released from such cells (1). One family of proteins that function as innate immune system recognition molecules is the pentraxins (2, 3). These molecules are characterized by their ancient origins, their cyclic pentameric (or in some cases hexameric) structure, and their calcium-dependent lectin-like binding to a variety of molecules. The two main members of the pentraxin family in man are C-reactive protein (CRP),3 a major acute phase reactant, and serum amyloid P component (SAP). SAP is a constitutive protein in human blood, where it is present at concentrations of ⬃40 ␮g/ml (4). In the mouse, SAP is a major acute-phase reactant (5). SAP is the precursor of tissue amyloid P component, where it may actually promote the formation of pathogenic amyloid deposits and prevent their degradation. SAP binds to microbial polysaccharides and matrix components through carbohydrate determinants, including heparin, 6-phosphorylated man-

*Veterans Affairs Medical Center; and Departments of †Medicine and ‡Molecular Genetics and Microbiology, University of New Mexico School of Medicine, Albuquerque, NM 87108 Received for publication August 11, 2000. Accepted for publication March 19, 2001. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by the Veterans Administration and by National Institutes of Health Grant AI28358. E.M. was supported by National Institutes of Health Grant SO6 GM-08139. 2 Address correspondence and reprint requests to Dr. Terry W. Du Clos, Veterans Affairs Medical Center, Research Service 151, Building T-12A, 1501 San Pedro Southeast, Albuquerque, NM 87108. E-mail address: [email protected] 3 Abbreviations used in this paper: CRP, C-reactive protein; SAP, serum amyloid P component; PMN, polymorphonuclear leukocytes; PIPLC, phosphatidylinositol-specific phospholipase C; PE-GAM, PE-F(ab⬘)2 goat anti-mouse IgG; GMCF, geometric mean channel fluorescence.

Copyright © 2001 by The American Association of Immunologists

nose, 3-sulfated saccharides, and the 4,6-cyclic pyruvate acetal of galactose (6, 7), in addition to its binding to amyloid fibrils. More recently, it has been recognized that SAP binds to chromatin and to DNA with high affinity (8). These molecules are major autoantigens in the human autoimmune disease systemic lupus erythematosus (9). It has recently been demonstrated that mice genetically deficient in SAP develop systemic lupus erythematosus with proliferative glomerulonephritis and characteristic autoantibodies (10). The mechanism of this effect is not known but is felt to involve alterations in the clearance of these nuclear Ags from the circulation. Consistent with this possibility, we previously demonstrated that SAP slows the clearance of chromatin from the circulation (11). The reduction in the rate of clearance was associated with increased uptake by the liver and decreased deposition in the kidneys. The existence of a receptor for SAP on mouse macrophages and human polymorphonuclear leukocytes (PMN) has been previously reported (12, 13). However, the nature of the receptor and its function were unknown. Becasue we recently determined that the receptors for the related pentraxin CRP are the IgG Fc receptors (Fc␥R) (14 –17), we tested whether Fc␥R would also bind SAP. When tested using transfected COS cells, all three classes of Fc␥R were found to bind SAP. One of the most important activities of Fc␥R is the mediation of phagocytosis (18). To determine whether SAP is capable of inducing phagocytosis, we used zymosan, a yeast to which SAP binds. SAP, as well as CRP, was shown to be an opsonin for yeast particles. Inhibition studies showed that opsonization by SAP and CRP used the same signaling pathway as IgG. SAP and CRP attached directly to beads also enhanced phagocytosis, indicating a direct interaction with phagocytic receptors. These results imply that SAP is an opsonin like CRP that could be an important part of the innate immune system with a role in the clearance of autoantigens. The ability of the pentraxins to interact with Fc␥R could also affect the presentation of autoantigens to T cells. 0022-1767/01/$02.00

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SAP BINDS TO Fc␥R AND INDUCES PHAGOCYTOSIS OF ZYMOSAN

Materials and Methods Reagents FITC-zymosan and rabbit anti-zymosan (zymosan-opsonizing reagent) were obtained from Molecular Probes (Eugene, OR). M-280 Dynabeads (tosyl-activated polystyrene beads coated with polyurethane) were purchased from Dynal Biotech (Lake Success, NY). Dynabeads were coated with CRP, SAP, or human IgG according to the manufacturer’s instructions using 5 ␮g protein/107 beads. Beads were blocked with Tris rather than albumin. Piceatannol, wortmannin, cytochalasin D, and phosphatidylinositol-specific phospholipase C (PIPLC) were purchased from Sigma (St. Louis, MO).

Antibodies Abs were purchased as follows: FITC anti-CD16 (mAb 3G8) was purchased from BD PharMingen (San Diego, CA), FITC anti-CD32 (mAb AT10) from Serotec (Raleigh, NC), anti-CD64 (mAb 32.2) from Medarex (Annandale, NJ), PE-anti-CD32 (mAb C1KM5) and PE-F(ab⬘)2 goat antimouse IgG (PE-GAM) from Caltag (Burlingame, CA), PE-anti-CD16 (mAb 3G8) from Calbiochem (San Diego, CA), PE anti-CD11b from Dako (Carpinteria, CA), and Alexa Fluor 488-F(ab⬘)2-GAM and Alexa Fluor 594-F(ab⬘)2 goat anti-human IgG from Molecular Probes. The hybridoma cell line producing mAb 2C10, a murine IgG1 anti-human CRP, was the generous gift of Dr. L. Potempa (ImmTech International, Evanston, IL) and was used as culture supernatant at 5 ␮g/ml. The mAb SAP-5, a murine IgG2a anti-human SAP, was purchased from Sigma and used as ascites at 5 ␮g/ml.

Isolation of CRP and SAP Human CRP was purified from pleural fluid by affinity chromatography and ion exchange chromatography as previously described (19). SAP was prepared as a side product of factor IX purification and generously provided to us by Dr. W. Kisiel (Department of Pathology, University of New Mexico, Albuquerque, NM). Briefly, SAP was copurified with coagulation factor IX from Proplex by immunoaffinity chromatography as described (20). SAP was then separated from factor IX by Q Sepharose Fast Flow chromatography. Minor contaminants were removed from SAP by anion exchange chromatography on a Mono Q column using FPLC (Pharmacia, Piscataway, NJ) with a 0.15– 0.5 M NaCl gradient in 20 mM Tris (pH 7.8) (21). SAP-biotin was prepared by coupling biotin to the carbohydrate moiety of SAP via a hydrazide linkage. SAP was oxidized with 10 mM sodium metaperiodate for 30 min in the dark. After the reaction was stopped with 15 mM glycerol, SAP was dialyzed overnight and treated with 5 mM biotin-LC-hydrazide (EZ-Link; Pierce, Rockford, IL) at pH 7 for 2 h. For flow cytometric binding studies, SAP-biotin was detected using PE-streptavidin (Caltag).

Cells Blood from normal volunteers was drawn into heparinized tubes. PMN were obtained by gradient separation using MonoPoly resolving medium (ICN Pharmaceuticals, Aurora, OH). In some cases, PMN were treated with 1 U/ml of PIPLC for 60 min at 37°. COS-7 cells were obtained from the American Type Culture Collection (Manassas, VA) and were maintained in DMEM with 10% FCS and 50 ␮g/ml gentamicin.

Cell transfections The human Fc␥RI cDNA clone, Fc␥RIaI in pRC/CMV, was obtained from Dr. R. P. Kimberly (University of Alabama, Birmingham, AL). The human Fc␥RIIA cDNA clone in the pcDSR␣296 transient transfection vector (22) was obtained from Dr. K. Moore (DNAX Research Institute, Palo Alto, CA). The human Fc␥RIIIb gene in pRC/CMV was obtained from Dr. E. Brown (University of California, San Francisco, CA). Cells were transfected using the GenePORTER transfection reagent from Gene Therapy Systems (San Diego, CA) as previously described (14). Mock-transfected cells received GenePORTER reagent only.

SAP binding assay Cells were washed twice in ice-cold PBS containing 0.05% azide and 0.1% globulin-free BSA and resuspended in this medium with SAP-biotin at the concentrations indicated. Cells were incubated for 1 h in the presence of SAP, then washed twice. Cells were then incubated for 30 min at 4°C with PE-streptavidin. Cells were washed twice and analyzed by flow cytometry. Fc␥RI expression was measured using mAb 32.2 and PE-GAM. Levels of Fc␥RII were determined by binding of FITC-AT10. Fc␥RIII was determined using PE-3G8 or FITC-3G8. The percentage of dead cells was de-

termined using 7-aminoactinomycin D according to the manufacturer’s directions (Molecular Probes).

Flow cytometry Cells were analyzed using a BD Biosciences (Mountain View, CA) FACSCalibur flow cytometer equipped with CellQuest software. The population analyzed was gated by forward and side scatter to exclude dead cells. A minimum of 30,000 cells was collected. For all measurements of SAP binding, the binding of PE-streptavidin has been subtracted. The results are expressed as the geometric mean channel fluorescence (GMCF).

Data analysis Dose-dependent binding curves of SAP to cells, as well as Scatchard plots, were generated using GraphPad Prism software (GraphPad, San Diego, CA). Binding of SAP was analyzed by nonlinear regression. Affinity constants were derived from the sum of least squares analysis by computer fitting, which is preferable to linear regression of the transformed data (23, 24). Scatchard graphs were created for graphic simplicity of data interpretation. Data shown are representative of at least two experiments. For phagocytosis assays, means were compared by two-tailed t tests.

Phagocytosis assays To measure phagocytosis, 2 ⫻ 106 FITC-zymosan were added to 5 ⫻ 105 PMN in RPMI 1640 supplemented with 5% FBS and 1 mM MnCl2. Tubes were centrifuged briefly (1 min at 200 ⫻ g) and incubated for 30 min at 37°C. Phagocytosis was scored visually on a Nikon fluorescent microscope (Nikon, Melville, NY). The fluorescence of the noningested yeast particles was quenched by the addition of trypan blue to a final concentration of 0.02%. The results are expressed as the phagocytic index (number of ingested yeast cells per 100 PMN). Similar phagocytosis assays were done using coated Dynabeads, except that, after incubation with PMN, uningested beads were stained with mAb to CRP or SAP and Alexa Fluor 488-GAM, or Alexa Fluor 594-anti-human IgG for IgG beads. Beads associated with phagocytic cells were determined by light microscopy and were scored as ingested or attached based on fluorescence.

Results

SAP binds to all three classes of Fc␥R in transfected COS cells We have recently demonstrated that the human leukocyte receptors for CRP are Fc␥RIIa and Fc␥RI (16, 25). Binding of CRP to each of these receptors can be demonstrated in transfected COS cells. Because SAP closely resembles CRP in both structural and binding characteristics, we decided to test whether SAP was capable of binding to Fc␥R as well. As shown in Fig. 1, SAP shows saturable binding to COS cells expressing either Fc␥RI or Fc␥RIIa compared with mock-transfected COS cells. These binding curves were determined using SAP-biotin detected with PE-streptavidin. Unmodified SAP was not used as an inhibitor of biotin-SAP due to the observed and previously reported tendency of SAP to undergo self-association at higher concentrations (26). Binding of unmodified SAP was demonstrated using a mAb to SAP and PE-GAM (data not shown). The calculated Kd values for these binding curves are 7.8 ⫻ 10⫺8 M for Fc␥RI and 1.4 ⫻ 10⫺7 M for Fc␥RIIa, which are close to the physiological concentration of SAP (⬃40 ␮g/ml or 3.5 ⫻ 10⫺7 M in serum). SAP binding to COS cells transfected with the human neutrophil receptor Fc␥RIIIb was also shown (Fig. 2). The binding curve is shown in Fig. 2A with a calculated Kd of 9.4 ⫻ 10⫺7 M. CRP does not bind to Fc␥RIIIbtransfected COS cells, which is consistent with our earlier results using peripheral blood leukocytes (data not shown). Fig. 2B shows two-color fluorescent analysis of Fc␥RIIIb-transfected COS cells after staining with PE-streptavidin for SAPbiotin binding and FITC-anti-CD16 for expression of Fc␥RIIIb. There was a strong association between the two markers with 28% double-positive cells, 57% double-negative cells, and ⬍10% of cells positive for either single marker.

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FIGURE 1. SAP binds to COS cells transfected with Fc␥RI or Fc␥RIIa. COS-7 cells were transfected with plasmids containing the Fc␥RI (A) or Fc␥RIIa (B) sequence using the GenePORTER transfection reagent. Mock-transfected cells reveived GenePORTER reagent only. Cells were incubated with increasing concentrations of SAP-biotin, and binding was detected by flow cytometry after staining with PE-streptavidin. The GMCF is shown for binding to transfected cells (total), mock-transfected cells, and the difference between the two curves (specific). Binding curves were generated by nonlinear regression. Scatchard plots of the specific binding are shown on the right.

SAP induces phagocytosis of zymosan by human PMN Although CRP has long been recognized as an opsonin, to our knowledge, the ability of SAP to enhance phagocytosis of microorganisms has not previously been demonstrated. Because phagocytosis is an important function of Fc␥R, we decided to test the effect of SAP on the phagocytosis of a model microbial particle. Previous studies have demonstrated the ability of both CRP and SAP to bind to zymosan, and we confirmed this (27). To determine whether SAP is an opsonin, human PMN were isolated and incubated with FITC-zymosan that had been treated with a saturating concentration of SAP (200 ␮g/ml) or rabbit IgG (zymosan-opsonizing reagent), and phagocytosis was determined by fluorescent microscopy (Fig. 3A). SAP and IgG treatment enhanced the uptake of FITC-zymosan from a phagocytic index of 25 for unopsonized zymosan to 61 for SAP ( p ⫽ 0.007) and 71 for IgG ( p ⫽ 0.010). In subsequent experiments, SAP was added to the incubation mixture containing PMN and FITC-zymosan. A dose-dependent enhancement of phagocytosis was observed when ⬎33 ␮g/ml SAP was added, with maximum effect at 100 ␮g/ml SAP (Fig. 3B). SAP had a greater effect when it was added to the incubation mixture

than when it was used to preopsonize FITC-zymosan. This is most likely due to a loss of SAP during washing, as SAP has a relatively low affinity for zymosan (3 ⫻ 10⫺5 M) (28). The addition of 10 ␮g/ml polymyxin B to bind endotoxin in the phagocytosis assay did not affect the results.

Effect of wortmannin and piceatannol on phagocytosis The signaling pathways required for phagocytosis by PMN through Fc␥R have been described (29). To determine whether enhanced uptake of SAP-zymosan was mediated by Fc␥R, we tested the ability of specific inhibitors of Fc␥R-mediated phagocytosis to block SAP and CRP opsonization. Wortmannin is a selective inhibitor of phosphatidylinositol-3 kinase and inhibits a number of Fc receptor-initiated responses including phagocytosis (29, 30). As shown in Fig. 4, treatment of PMN with wortmannin (10 nM) reduced the phagocytosis of SAP (72 ⫾ 12% decreased; p ⫽ 0.008), CRP (73 ⫾ 3% decreased; p ⬍ 0.001), and IgGopsonized zymosan (62 ⫾ 11% decreased; p ⫽ 0.012) to the level

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FIGURE 2. SAP binds to COS cells transfected with Fc␥RIIIb. COS-7 cells were transfected with a plasmid containing the Fc␥RIIIb sequence using the GenePORTER transfection reagent. Mock-treanfected cells received GenePORTER reagent only. A, Cells were incubated with increasing concentrations of SAP-biotin, and binding was detected by flow cytometry after staining with PE-streptavidin. The GMCF is shown for binding to transfected cells (total), mock-transfected cells, and the difference between the two curves (specific). Binding curves were generated by nonlinear regression. Scatchard plots of the specific binding are shown on the right. B, COS cells transfected with Fc␥RIIIb or mock transfected were analyzed by flow cytometry for expression of Fc␥RIIIb using FITC-anti-CD16 and for binding of SAP-biotin (150 ␮g/ml) using PE-streptavidin.

observed with unopsonized zymosan. Piceatannol is a specific inhibitor of the tyrosine kinase Syk, which is required for Fc␥Rmediated phagocytosis but not for uptake of latex beads, bacteria, or complement-opsonized yeast (31, 32). As shown in Fig. 5, treatment of PMN with piceatannol (2–10 ␮M) also inhibited the phagocytosis of SAP-opsonized (33 ⫾ 10% decreased; p ⫽ 0.051) and CRP-opsonized (68 ⫾ 9% decreased; p ⫽ 0.005) zymosan to the level observed with unopsonized zymosan. IgG induced phagocytosis of zymosan was reduced by 52 ⫾ 9% ( p ⫽ 0.021). Uptake of unopsonized zymosan was not significantly decreased by either inhibitor. These results indicate that both SAP and CRP enhanced

phagocytosis of zymosan requires phosphatidylinositol-3 kinase and Syk. This is consistent with Fc␥R-mediated phagocytosis. PIPLC treatment of human PMN results in decreased phagocytosis of FITC-zymosan We previously reported that Fc␥RIIa is the primary CRP receptor on human PMN. PMN express ⬃10 times more Fc␥RIIIb than Fc␥RIIa. Because SAP binds to Fc␥RIIIb as well as Fc␥RIIa, we wished to determine the role of Fc␥RIIIb in SAP opsonization. Fc␥RIIIb is a GPI-anchored protein and can therefore be removed by treatment of cells with PIPLC. The results in Fig. 6 demonstrate

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FIGURE 5. Phagocytosis of SAP-, CRP-, or IgG-opsonized zymosan is inhibited by piceatannol. PMN were treated with 2 or 10 ␮M piceatannol or 1% ethanol (vehicle control) for 30 min at room termperature. FITCzymosan was added to PMN at a ration of 4:1 together with 100 ␮g/ml SAP or CRP anti-zymosan Ab. Reaction mixtures were centrifuged to enhance contact and incubated for 30 min at 37°C. Trypan blue was added to quench the fluorescence of external particles, and phagocytosis was scored by fluorescent microscopy. Results are expressed as the number of FITCzymosan ingested per 100 PMN (phagocytic index). The results shown are from a representative experiment (of four). FIGURE 3. SAP opsonizes zymosan for phagocytosis by human PMN. A, FITC-zymosan was incubated with 200 ␮g/ml of SAP or rabbit antizymosan Ab for 30 min at 37°C and then washed once with HBSS. Opsonized zymosan was then added to PMN at a ratio of 4:1. PMN and FITC-zymosan were centrifuged to enhance contact and incubated for 30 min at 37°C. Trypan blue was added to quench the fluorescence of external particles, and phagocytosis was scored by fluorescent microscopy. Results are expressed as the number of FITC-zymosan ingested per 100 PMN (phagocytic index). The results shown are the means ⫾ SEM of three experiments. B, FITC-zymosan was added to PMN at a ratio of 4:1 together with increasing concentrations of SAP. Reaction mixtures were centrifuged to enhance contact and incubated for 30 min at 37°C. Trypan blue was added to quench the fluorescence of external particles, and phagocytosis was scored by fluorescence microscopy. Results are expressed as the number of FITC-zymosan ingested per 100 PMN (phagocytic index). The results shown are the means ⫾ SEM of two experiments.

Phagocytosis of beads Because PMN can ingest unopsonized zymosan through CD11b/ CD18, it was possible that SAP and CRP were providing an activating signal for uptake of zymosan through these receptors (33) rather than directly binding to Fc␥R to promote ingestion of zymosan. Therefore, we also tested the ability of SAP and CRP to opsonize Dynabeads, which are not bound or ingested by PMN in the absence of opsonins (phagocytic and attachment indexes ⬍10). Dynabeads were coated with CRP, SAP, and IgG and tested for

reduced phagocytosis by SAP (44 ⫾ 12% decreased; p ⫽ 0.039) and IgG (43 ⫾ 8% decreased; p ⫽ 0.013) using PIPLC-treated PMN. Uptake of CRP-opsonized zymosan was not affected (16 ⫾ 4% decreased). B shows the selective removal of Fc␥RIIIb and not Fc␥RIIa or CD11b by the PIPLC treatment.

FIGURE 4. Phagocytosis of SAP-, CRP-, or IgG-opsonized zymosan is inhibited by wortmannin. PMN were treated with 10 nM wortmannin for 30 min at room termperature. FITC-zymosan was added to PMN at a ration of 4:1 together with 100 ␮g/ml SAP or CRP anti-zymosan Ab. Reaction mixtures were centrifuged to enhance contact and incubated for 30 min at 37°C. Trypan blue was added to quench the fluorescence of external particles, and phagocytosis was scored by fluorescent microscopy. Results are expressed as the number of FITC-zymosan ingested per 100 PMN (phagocytic index). The results shown are from a representative experiment (of four).

FIGURE 6. PIPLC treatment of PMN decreases phagocytosis of SAPopsonized zymosan. PMN were treated with 1 U/ml PIPLC to remove GPI-anchored membrane proteins. A, FITC-zymosan was added to PMN at a ratio of 4:1 together with 100 ␮g/ml SAP or CRP or anti-zymosan Ab. Reaction mixtures were centrifuged to enhance contact and incubated for 30 min at 37°C. Trypan blue was added to quench the fluorescence of external particles, and phagocytosis was scored by fluorescent microscopy. Results are expressed as the number of FITC-zymosan ingested per 100 PMN (phagocytic index). The results shown are from a representative experiment (of four). B, Staining with mAb to Fc␥RIII (CD16), Fc␥RII (CD32), and CD11b shows selective removal of Fc␥RIII, which is GPIanchored, and no change in expression of Fc␥RII or CD11b, which are transmembrane proteins.

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phagocytosis by PMN. All three opsonins promoted both adherence and ingestion of these inert beads ( p ⬍ 0.05 for each opsonin compared with controls), providing further evidence for a direct interaction between SAP or CRP and phagocytic receptors. Dynabeads coupled to either human serum albumin or BSA were phagocytosed less efficiently than Tris-blocked beads.

Discussion The major new findings reported here are first that SAP interacts with all three classes of human Fc␥R and, second, that SAP effectively opsonizes zymosan and polystyrene beads for phagocytosis by human PMN. The importance of Fc␥R in SAP-mediated phagocytosis was shown by experiments using inhibitors of Fc␥R signaling. Taken together, these results suggest that SAP, like CRP, interacts with Fc␥R to mediate phagocytosis. These findings establish a potentially important role for SAP in the innate immune system. The interaction of SAP with Fc␥R on transfected cells was found to be specific and saturable with binding affinities within the physiological range of SAP. SAP levels vary from ⬃20 –50 ␮g/ml in normal individuals, with males having slightly higher levels than females (34). The binding of SAP to human PMN was previously demonstrated by Landsmann et al. (13). These investigators reported that SAP bound to human PMN with high affinity and that CRP and SAP competed for binding. The binding of SAP to PMN showed heterogeneity of affinities consistent with multiple binding sites. The highest affinity sites had a Kd of 5 ⫻ 10⫺8 M. Although our studies cannot precisely determine binding affinities due to the use of flow cytometry, we detected binding of similar affinity to Fc␥RI and Fc␥RIIa. Binding of SAP to Fc␥RIIIb was of lower affinity. The finding of multiple binding sites on PMN with different affinity is consistent with binding to Fc␥RII and Fc␥RIII, as Fc␥RI is not present on resting PMN. Siripont et al. (12) also reported the presence of a high affinity receptor for mouse SAP on mouse macrophages. However, the binding appeared to occur in a calcium-dependent manner. Furthermore, the binding was inhibited by mannose and is therefore unlikely to be related to the findings described here. Because phagocytosis is an important activity mediated by Fc␥R (18), we tested the ability of SAP to opsonize zymosan for phagocytosis by PMN. To our knowledge, this is the first description of the opsonic activity of SAP. The phagocytosis of a fungal cell suggests that SAP could be involved in protection of the host from infection similar to CRP, which has been demonstrated to protect mice from infection with Streptococcus pneumoniae (35, 36). Experiments designed to examine this possibility are currently in progress. Selective inhibitors of Fc␥R-mediated phagocytosis were used to determine whether SAP opsonization was mediated through binding to Fc␥R. Both wortmannin, a phosphatidylinositol-3 kinase inhibitor, and piceatannol, a Syk kinase inhibitor, decreased the uptake of SAP- and CRP-opsonized zymosan to the level of unopsonized zymosan. These inhibitors block FcR signaling pathways including Fc␥R-mediated phagocytosis (29). Syk is required for phagocytosis of IgG-opsonized ligands but not complementopsonized zymosan or bacteria (31, 32). Thus, the finding that treatment of PMN with these inhibitors prevented opsonization by SAP and CRP is consistent with pentraxin binding to and stimulating phagocytosis through Fc␥R. An alternative mechanism by which SAP and CRP could enhance phagocytosis would be through activation of other receptors for zymosan. PMN phagocytose unopsonized zymosan through CD11b/CD18 (37). Attachment of monocyte-derived macrophages to SAP-coated wells was previously found to increase uptake of

erythrocytes opsonized with complement (C3b or iC3b), which is mediated through complement receptor type 1 and CD11b/CD18. Therefore, to confirm that SAP and CRP are directly opsonic, they were conjugated to polystyrene beads. The unopsonized beads were poorly recognized by PMN with ⬍10 beads attached or ingested by 100 PMN. However, SAP, CRP, and IgG-conjugated beads readily attached to and were ingested by PMN. Thus, CRP and SAP are capable of mediating phagocytosis in the absence of surface ligands for other receptors. The interaction of SAP with Fc␥R may be especially relevant to the clearance of self Ags. The interaction of SAP with chromatin and DNA that are released from apoptotic and necrotic cells has been described (38, 39). SAP may play an important role in the handling of the potent autoantigens that may lead to immunization on one hand and, on the other hand, tissue deposition leading to immune complex-mediated inflammation. It is still unclear whether SAP will cause a proinflammatory response associated with phagocytosis. It is of interest to compare the binding of SAP to Fc␥R with the recently described interaction of CRP with Fc␥R. The two molecules are closely related, and both are conserved molecules that preceded the development of Ig. It is likely that these two molecules evolved early on as primitive defense molecules that recognized pathogens through pattern recognition of carbohydrates and other ligands on pathogens. We have speculated that the Fc receptors may have evolved first as pentraxin receptors and, with development of Ig, were used by Ig as surface receptors. Although both CRP and SAP have now been shown to bind to phagocytic cells through Fc␥R, they appear to differ in their affinity for the individual Fc␥R. CRP binds to both Fc␥RI and Fc␥RIIa, but no binding to Fc␥RIII was detected. In contrast, SAP binds to all three classes of Fc␥R. Consistent with the binding data, PIPLC treatment of PMN to remove GPI-anchored proteins including Fc␥RIII decreased phagocytosis of SAP-opsonized zymosan but had little effect on uptake of CRP-opsonized zymosan. Recently, we have characterized the binding of SAP to Fc␥R in the mouse through the use of Fc␥R-deficient mouse strains (27). The results in the mouse are similar to those using human Fc␥R: both pentraxins stimulate phagocytosis through Fc␥RI and SAP, but CRP does not induce phagocytosis through Fc␥RIII. The findings reported here suggest that SAP is an important mediator of phagocytosis in vitro at physiological concentrations. Thus, it is likely that SAP may function as an opsonin for natural ligands, which include damaged cells, chromatin, and DNA, as

FIGURE 7. SAP, CRP, and IgG enhance attachment and ingestion of Dynabeads by PMN. Dynabeads were blocked with Tris (buffer) or coated with SAP, CRP, or IgG and added to PMN at a ratio of 10:1. Reaction mixtures were centrifuged to enhance contact and incubated for 30 min at 37°C. Beads that were not internalized were stained with fluorescent Abs. Phagocytosis and attachment of beads was scored by fluorescent microscopy. Results are expressed as the number of beads ingested or attached per 100 PMN (phagocytic or attachment index). The results shown are means ⫾ SEM of three experiments.

The Journal of Immunology well as for foreign particles such as microorganisms. Because SAP has been shown to activate complement and enhance phagocytosis, it may an important role in host defense and in clearance of autoantigens.

Acknowledgments We thank Dr. Walter Kisiel for providing SAP and Michael A. Volzer and Carol A. Morris for expert technical support.

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