The Journal of Immunology

Complement Receptor of the Ig Superfamily Enhances Complement-Mediated in a Subpopulation of Tissue Resident Macrophages

Nick N. Gorgani,1* Jeannie Q. He,1* Kenneth J. Katschke, Jr.,* Karim Y. Helmy,* Hongkang Xi,* Micah Steffek,† Philip E. Hass,‡ and Menno van Lookeren Campagne2*

An important function of the complement cascade is to coat self and foreign particles with C3- that serve as ligands for phagocytic receptors. Although tissue resident macrophages play an important role in complement-mediated clearance, the re- ceptors coordinating this process have not been well characterized. In the present study, we identified a subpopulation of resident peritoneal macrophages characterized by high expression of of the Ig superfamily (CRIg), a recently dis- covered complement C3 receptor. Macrophages expressing CRIg showed significantly increased binding and subsequent inter- nalization of complement-opsonized particles compared with CRIg negative macrophages. CRIg internalized monovalent ligands ␤ 2؉ 2؉ and was able to bind complement-opsonized targets in the absence of Ca and Mg , which differs from the 2-integrin CR3 that requires divalent cations and polyvalent ligands for activation of the receptor. Although CRIg dominated in immediate binding of complement-coated particles, CRIg and CR3 contributed independently to subsequent particle phagocytosis. CRIg thus iden- tifies a subset of tissue resident macrophages capable of increased phagocytosis of complement C3-coated particles, a function critical for immune clearance. The Journal of Immunology, 2008, 181: 7902–7908.

hagocytosis, an important component of host defense and and conformational changes, two processes that require activat- a primary function of macrophages, is facilitated by op- ing stimuli (9, 10). Upon activation of phagocytes with phorbol P sonization, a process by which serum components tag esters (11) or ␤ glucan (12, 13), cell surface expression and pathogens and immune complexes for recognition by activation state of CR3 are increased, enabling CR3-mediated and macrophages (1). Triggering of the classical, lectin, and alter- phagocytosis. Similarly, inflammatory macrophages freshly re- native pathways of complement activates the central component cruited from the circulation to peritoneal cavity use CR3 for C3 resulting in covalent binding of to a particle with the phagocytosis (14, 15). Although tissue resident macrophages release of . C3b is rapidly degraded to iC3b by the action of are thought to play an important role in both initiation and factor I and cofactors and further degraded to C3c, C3d, and C3dg resolution of inflammation (16–18), CR3 does not act as a po- (2, 3). Binding of the C3dg and C3d fragments to CR2 on B cells tent phagocytic CR on these cells (19). leads to enhanced Ab production (4), whereas binding of C3 pro- Recently, we identified a novel CR of the Ig superfamily teins to complement receptors (CRs);3 on macrophages results in (CRIg) that is highly expressed on Kupffer cells (20, 21). CRIg phagocytosis and clearance of the particle (5–8). is required for rapid sequestration of -borne Listeria CR3, a transmembrane heterodimer composed of two integrin monocytogenes and Staphylococcus aureus in a C3-dependent ␣ ␤ subunits (CD11b or M and CD18 or 2), is a well established manner, thereby limiting systemic bacteremia and promoting CR for the dominant C3 cleavage product, iC3b, and in mice is survival of the host. In this study, we show that CRIg is re- present on all myeloid cells and a subset of NK cells (7). CR3- quired for efficient complement-mediated phagocytosis by non- mediated phagocytosis is dependent on receptor redistribution activated as well as activated resident peritoneal macrophages (RPMs). Thus, CRIg identifies a subset of tissue macrophages that is capable of integrin-independent rapid recognition and *Department of Immunology, † Engineering, and ‡Protein Chemistry, Genen- phagocytosis of iC3b-opsonized particles in the absence or tech, South San Francisco, CA 94080 presence of inflammatory stimuli, a feature of importance for Received for publication December 24, 2007. Accepted for publication September tissue homeostasis and host defense. 30, 2008. 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 Materials and Methods with 18 U.S.C. Section 1734 solely to indicate this fact. Abs, proteins, and dyes 1 N.N.G. and J.Q.H. contributed equally to the work. 2 Non-blocking mAbs against CRIg were generated as described (20). Address correspondence and reprint requests to Menno van Lookeren Campagne, Blocking Abs (clone 2H1 and 14G8; Genentech) were generated by im- Genentech, 1 DNA Way, South San Francisco, CA 94080. E-mail address: menno@.com munizing CRIg-deficient mice with CRIg-Fc fusion protein generated as described (20). Phycoerytherin-labeled anti-F4/80 Ab (F4/80-PE) was pur- 3 Abbreviations used in this paper: CR, complement receptor; CRIg, CR of the Ig chased from Caltag Laboratories. Unless indicated, all other Abs were superfamily; RPM, resident peritoneal macrophage; naRPM, nonadherent RPM; ad- purchased from BD Biosciences. C4b was purchased from Complement RPM, adherent RPM; 7-AAD, 7-aminoactinomycin D; HGDMEM, high glucose DMEM; E-IgM, IgM-coated erythrocyte; EAC, complement-opsonized E-IgM; Technologies. Human C3, C3b, and iC3b (used in Fig. 2) were obtained SRBC, sheep RBC; Crry, CR-related gene Y; MFI, mean fluorescent intensity; DAPI, from fresh serum and purified as described (20). Murine C3 was purified 4Ј,6-diamidino-2-phenylindole. from fresh mouse serum using the same method. Murine C3b was gener- ated by cleavage of purified C3 by trypsin as described elsewhere (22). Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 Murine iC3b was purified from pooled murine ascites obtained from the www.jimmunol.org The Journal of Immunology 7903 i.p. cavity following implantation of various hybridomas. Ascites was buff- ered with 25 mM Tris (pH 7.5), 5 mM EDTA, and 100 mM NaCl. PEG 6000 was added to the ascites to a final concentration of 10% and contin- uously mixed for 1 h. This solution was centrifuged at 20,000 ϫ g for 15 min. The supernatant was decanted, and the pellet was re-suspended in 50 mM Tris (pH 8.6) and 5 mM EDTA and loaded over a protein G column to deplete the IgG from the solution. The flow through was applied directly onto a mono Q (10 ϫ 100; GE Healthcare), which was eluted with a gradient from 0 to 0.7 M NaCl over 20-column volumes. The murine iC3b was isolated and loaded separately over a Superdex 200 (16/60; GE Health- care) at 7 cm/h in 25 mM Tris (pH 8.0), 150 mM NaCl, and 5 mM EDTA. The resulting fractions were analyzed by SDS-PAGE, and identity was confirmed by Edman degradation. Abs and proteins were labeled with Alexa Fluor 555 (A555), Alexa Fluor 488 (A488), or Alexa Fluor 647 (A647) according to manufacturer’s instruction (Invitrogen). CRIg-ECD was generated by expression of the extracellular domain of murine CRIg in Chinese hamster ovary cells and purified as described (20). All other reagents are from Sigma-Aldrich un- less noted. Animals and flow cytometry analysis of resident peritoneal macrophages All animals were held under sterile pathogen free conditions and animal experiments were approved by the Institutional Animal Care and Use Com- mittee of Genentech. Six to 7-wk old C57BL/6J mice and AKR mice were purchased from Jackson ImmunoResearch Laboratories. C3-deficient mice have been described (23). Resident peritoneal cells were lavaged with PBS containing 2 mM EDTA, resuspended in PBS containing 1% BSA, and incubated on ice with anti-mouse CD16/32 and purified rat IgG2a and IgG2b to block Fc receptors followed by A647-conjugated anti-CRIg Ab (clone 17C9), PE-conjugated Ab to F4/80, anti-CR1/CR2 (clone 7G6), anti-CR3 (CD11b, clone M1/70), anti-CD18, anti-CR4 (CD11c, clone HL3), or anti-CR-related gene Y (Crry; clone 1F2). After incubation with 7-amino-actinomycin D (7-AAD) (Molecular Probes), cells were analyzed by flow cytometry on a FACSCalibur (BD Biosciences). For immunohis- tochemistry of CRIg, CRIgϩ F4/80ϩ and CRIgϪ F4/80ϩ nonadherent RPMs (naRPMs) were sorted (FACSAria 1; BD Biosciences) and cultured in Lab-Tek II chamber slides (Nunc) in DMEM (High glucose DMEM (HGDMEM); Cellgro) containing 10% heat inactivated FBS (HyClone), FIGURE 1. CRIg and CR3 are the only phagocytic complement C3 4.5 mg/ml glucose, 10 mM HEPES, 10 mM glutamine, 100 U/ml penicil- receptors expressed on RPMs. A, Flow cytometry analysis of CRIg expres- lin, and 100 ␮g/ml streptomycin (Invitrogen) (HGDMEM-10). Cells were ϩ ϩ Ϫ sion on F4/80 RPMs. B and C, CRIg and CRIg RPMs express similar fixed with acetone/ethanol solution (75/25, v/v) at Ϫ20°C for 10 min, blocked for FcR-mediated binding as indicated above, and stained for CRIg levels of CD11b, CD18, and Crry, but lack expression of CR1, CR2, and as described (20). CD11c. Shaded areas represent specific staining, solid lines represent iso- type Ab staining. Histograms are representative of three independent ex- Binding and internalization of complement proteins by adherent periments. D, CRIg (red) is expressed on RPMs. CRIgϩ and CRIgϪ RPMs RPMs (adRPMs) were FACS sorted and cultured for 7 days before immunostaining. Blue indicates nuclear staining with DAPI. Scale bar (D) ϭ 10 ␮m. For microscopic evaluation of iC3b, C3b, and C3 internalization, cells were incubated in Lab-Tech II chamber slides (Nunc) for1hat37°C in HGD- MEM, nonadherent cells were removed and, after overnight, culture cells were stained with an A555-labeled anti-CRIg Ab (clone 14G6), incubated naRPMs were resuspended in PBS/0.5% BSA with either 10 mM EDTA or with A488-labeled complement proteins, and internalization visualized by ϩ ϩ 0.15 mM Ca2 /1 mM Mg2 and incubated with EACs for 30 min at 37°C. microscopy. To determine the binding and phagocytosis of EACs to adRPMs, 1ϫ106 Binding and phagocytosis of sheep RBC (SRBC) peritoneal cells were placed in 6-well tissue culture plates (Costar) or in chamber slides in HGDMEM-10, nonadherent cells were removed after 1 h SRBCs (5 ϫ 108/ml; Colorado Serum) were labeled with rat IgM-A488 incubation at 37°C, and adherent cells were cultured overnight. Where anti-Forsman Ab (clone TIB-123, 5 ␮g/ml; ATCC) for 30 min at 22°C indicated, adRPMs were activated with 1 ␮g/ml LPS (Escherichia coli (IgM-coated erythrocyte; E-IgM). 2.5 ϫ 108 E-IgM were incubated with 1 strain O26:B6; Sigma-Aldrich) for 24 h. Cells were placed on ice, washed ml Veronal Buffer (BioWhittaker) containing 10% serum from C5-defi- 2ϫ with cold PBS, and FcRs were blocked with cold HGDMEM/5 ␮g/ml cient AKR/J mice (The Jackson Laboratory), 0.15 mM CaCl, 1 mM MgCl, anti-CD16/32 (BD Biosciences), 20 ␮g/ml anti-ragweed mIgG2a and and 0.1% gelatin for 30 min at 37°C to generate complement-opsonized mIgG2b (Genentech) for 10 min on ice. FcR blocking solution was then E-IgM (EAC). Serum from AKR/J mice was used to prevent lysis of the aspirated and 50 ␮g/ml anti-CRIg (14G8), anti-CD11b (clone M1/70), or SRBCs. For analysis of C3 fragments present on the EACs, EACs were isotype control Abs was added for 10 min on ice to block CRs. A total of then lysed in 1% NP40 at 1E8c/ml, an equal volume of 2ϫ gel loading 2 ϫ 107 EACs in cold HGDMEM was added and binding synchronized by buffer plus 10% ␤-mercaptaethanol was added to yield a final cell lysate spinning at 500 rpm for 1 min at 4°C. Cells were placed at 37°C for concentration of 5E7c/ml. 5E5 EACs in a volume of 10 ␮l were loaded per indicated times and phagocytosis was stopped by placing cells on ice. To well in 8% SDS-PAGE. C3 proteins were detected using 1 ␮g/ml goat monitor phagocytosis by flow cytometry, cells were washed in PBS/10 mM anti-mouse C3 (MP Biomedicals) and rabbit anti-goat Ab (Thermo Fisher EDTA, scraped off, transferred to chilled FACS tubes, washed 1ϫ with Scientific). PBS/0.5% BSA, and stained with F4/80-PE and anti-CRIg A647 (clone Binding experiments were performed on freshly lavaged, naRPMs re- 25C5) for 25 min. Extracellular SRBCs were lysed with 1 ml of ACK lysis suspended in PBS/0.5% BSA. Cells were placed on ice and FcRs were buffer (82 mM NH4Cl, 5 mM KHCO3, and 50 nM EDTA) for 2 min on ice, blocked as described above plus 25 ␮g/ml anti-CRIg (clone 14G8), anti- washed 2ϫ with PBS, and then incubated with 30 ␮g/ml anti-A488 Ab CD11b (clone M1/70), or isotype control (anti-ragweed; Genentech) block- (Invitrogen) to quench fluorescence of A488-conjugated EACs bound to ing Abs. EACs were added to the naRPMs in a 20:1 ratio for 30 min at the adRPM cell surface. Cells were analyzed on FACSCalibur, 1 ␮g/ml 37°C and the amount of EACs bound determined by flow cytometry as propidium iodide was added to exclude dead cells, and data analyzed with described below. To determine the effect of divalent cations on binding, FlowJo software (TreeStar). 7904 CRIg AND CR3 IN COMPLEMENT-MEDIATED PHAGOCYTOSIS

FIGURE 3. CRIg is sufficient for binding of iC3b-opsonized particles to naRPMs. A, left panel, Flow cytometry of complement C3 proteins found on EACs opsonized with serum from C3-deficient mice or C5-deficient AKR mice. Solid lines represent isotype control Ab staining, shaded lines represent staining for C3. Right panel, Western blot analysis of EACs FIGURE 2. CRIg, but not CR3, binds and internalizes C3b and iC3b. A, opsonized with serum from AKR mice or with serum from C3-deficient A488-conjugated purified iC3b, C3 and C4b were added at different con- mice. Purified mouse C3 proteins (right lanes) are used as reference. B, centrations to suspensions of naRPMs and mean fluorescent intensity naRPMs were mixed with A488-labeled EACs on ice or incubated for (MFI) units of the bound protein were analyzed by flow cytometry. Results various times at 37°C and then stained with a non-blocking Ab to CRIg. ϩ shown are from one experiment representative of three. B, Binding of iC3b The MFI of A488 was determined by flow cytometry. C, left panel, CRIg Ϫ to CRIgϩ naRPMs is significantly reduced in the presence of CRIg-block- and CRIg naRPMs were incubated for 30 min at 37°C with control or ing Ab (CRIg Ab) and extracellular domain of murine CRIg (CRIg-ECD), CR3 and/or CRIg blocking Abs and A488-labeled EACs. The amount of ϩ Ϫ but remains unaltered in the presence of CR3 blocking Ab (CR3 Ab). EACs bound to CRIg and CRIg naRPMs was then determined by stain- p Ͻ 0.001. C, Colocal- ing the cells with a nonblocking Ab to CRIg and measuring the MFI of ,ء .Results are expressed as mean Ϯ SD, n ϭ 3 ization of CRIg and C3 proteins. Cultured adRPMs were incubated with A488 using flow cytometry. Right panel, naRPMs were treated as de- A488-conjugated iC3b, C3b, and C3 (green). Cells were then stained for scribed above but incubated with A488-labeled EACs in the presence of 20 ;p Ͻ 0.05 ء ;CRIg (red) and nuclei were visualized by DAPI staining (blue). Inset, Co- mM EDTA. Results are expressed as mean Ϯ SD, n ϭ 3 .p Ͻ 0.001 ءء localization of iC3b and CRIg in the perinuclear region as well as in the tips of the pseudopodia. Results shown are from one experiment represen- tative of three. CRIgϪ adRPMs are indicated with arrows. Scale bar (C) ϭ Sequential images were acquired with a spacing of 0.2 microns along the 10 ␮m. z-axis and then deconvolved using softWoRx software (Applied Precision). Statistical analysis For immunufluorescence, adRPMs with EACs were washed with PBS/ Statistical analysis was performed using JMP software (JMP Release:

0.15 mM CaCl2/1 mM MgCl2, FcRs were blocked for 10 min and the 5.1.2; SAS Institute). All p values are calculated with an unpaired Student’s blocking solution was then removed, and, finally, cells were stained with 3 t test assuming unequal variance. ␮g/ml A555-conjugated CRIg (clone 14G6) Abs for 15 min on ice. In some experiments, nonfluorescent EACs and 3 ␮g/ml anti-CD11b-FITC was Results used. Cells were washed and extracellular EACs lysed with ACK buffer for CRIg is expressed on a subpopulation of RPMs 2 min. In experiments where A488-conjugated EACs were used, the non- ␣ ␤ internalized EAC membrane was quenched with an anti-A488 Ab. adRPMs Both CRIg and CR3 (or M 2) are receptors for iC3b present on were fixed with 2% paraformaldehyde (EM Sciences) in PBS for 20 min serum-opsonized pathogens. To determine the relative contribution Ј and cover-slipped with VectaShield with 4 ,6-diamidino-2-phenylindole of CRIg and CR3 to complement-mediated phagocytosis, we fo- (DAPI) (Vector Laboratories). cused our studies on RPMs that express high levels of CR3. CRIg Fluorescence and deconvolution microscopy was coexpressed with CR3 on ϳ30% of the total population of Images were acquired on a Nikon Eclipse TE300 inverted microscope us- RPMs (Fig. 1A) in line with a previous report (24). Next to ex- ing a ϫ60 objective, a Zeiss LSM 510 confocal microscope using a 60ϫ pression on the cell surface, CRIg was also localized on an intra- objective, or an Olympus BX61 upright microscope using a ϫ60 objective. cellular pool of recycling endosomes, as described in our previous The Journal of Immunology 7905

take of C3 in CRIgϩ adRPMs was observed and likely indicates that some of the C3 was hydrolyzed and subsequently internalized through CRIg. CRIgϪ adRPMs, identified by nuclear staining only (Fig. 2C, arrows), did not internalize any of the C3 proteins. These results illustrate that CRIg is able to bind and internalize mono- meric ligands while CR3 is not, confirming the requirement of a multivalent ligand for CR3-mediated binding and internalization of target particles (9, 10, 28). CRIg and CR3 differ in their requirement for binding to complement-opsonized particles To distinguish the contribution of CRIg and CR3 to binding and phagocytosis of particles coated with multivalent C3 proteins, IgM-opsonized SRBC were incubated with C5-deficient serum, obtained from AKR/J mice, to generate complement C3-coated SRBC (EACs). Flow cytometry analysis indicated deposition of C3 proteins on the surface of EACs that were incubated with C5- deficient serum but not on EACs incubated with C3-deficient se- rum (Fig. 3A, left panel). The major C3 protein coated on the surface of these particles was in the form of iC3b as illustrated by Western blot analysis using purified C3 proteins as a reference FIGURE 4. CRIg and CR3 localize to phagosomes. adRPMs cultured (Fig. 3A, right panel). Binding experiments were conducted using overnight were stained with an anti-CR3 (green) or anti-CRIg (red) Ab naRPMs. Under the conditions used and in line with a previous before (A), or following (B), incubation with EACs for 30 min. Blue in- report (29), all A488-conjugated EACs bound to the surface of the dicates nuclear staining with DAPI. Arrows indicate accumulation of CRIg naRPMs were not internalized as shown by the reduction of flu- and CR3 in the phagosomes surrounding the EACs. Scale bar ϭ 20 ␮m. orescence to baseline values following lysis of these EACs and quenching of the extracellular fluorescence with anti-A488 Abs (results not shown). EACs incubated with C3-deficient serum did ϩ Ϫ study (20). CRIg and CRIg macrophages did not differ in shape not bind to naRPMs (results not shown) indicating that all binding or granularity as indicated by similar forward- and side-scatter was mediated through complement C3 receptors. At 4°C, CRIgϩ profiles (Fig. 1B). CR3 and Crry, the latter acting as a regulator of naRPMs showed significantly increased binding of EACs com- ϩ complement activation, were uniformly expressed on CRIg and pared with CRIgϪ naRPMs (Fig. 3B). Although both CRIgϪ and Ϫ ϩ Ϫ CRIg RPMs (Fig. 1C) (25). Neither CRIg nor CRIg RPMs CRIgϩ naRPMs increased their binding to EACs with time at expressed CR1, CR2, or CD11c, the ␣-chain of CR4 in accordance 37°C, the amount of complement-opsonized particles bound to ϩ with a previous report (26). To establish whether CRIg and CRIgϩ naRPMs remained significantly greater than the number of Ϫ CRIg RPMs represent different maturation stages of peripheral particles bound to CRIgϪ naRPMs. blood-derived entering the peritoneal cavity from the CRIg and CR3 blocking Abs were subsequently used to deter- periphery, RPMs were FACS-sorted, cultured separately for 1 wk, mine the relative contribution of CRIg vs CR3 on naRPMs to ϩ permeabilized, and stained for CRIg. CRIg RPMs maintained particle binding after incubation for 30 min at 37°C. In CRIgϩ CRIg expression on the cell surface and in the cytoplasm for at naRPMs, blocking of CRIg or CR3 reduced the binding of EACs Ϫ least 7 days in culture, whereas CRIg RPMs neither expressed by 30 and 50%, respectively, whereas blocking both receptors si- cell surface nor cytoplasmic CRIg (Fig. 1D). Thus, CRIg is stably multaneously reduced particle binding to baseline levels (Fig. 3C, expressed on a subpopulation of tissue resident macrophages that left panel). In the CRIgϪ population of naRPMs, binding was fully also expresses CR3. mediated through CR3. Next, binding studies were performed in the absence of divalent cations to exclude binding through CR3 CRIg is required for binding and internalization of monomeric which is Mg2ϩ-dependent (9). When chelating both Ca2ϩ and C3-derived proteins Mg2ϩ with EDTA, binding of EACs to CRIgϩ naRPMs was re- Since iC3b is the predominant C3 fragment found on complement- duced by 60% while negligible binding to CRIgϪ naRPMs was opsonized particles (27), we determined the contribution of CRIg detected. In CRIgϩ naRPMs, Ca2ϩ/Mg2ϩ-independent binding ac- vs CR3 in binding and internalization of soluble iC3b. For binding tivity was entirely mediated through CRIg as CRIg blocking Abs studies, freshly lavaged, naRPM were used. When incubated at completely abolished EACs binding to naRPMs (Fig. 3C, right 4°C, iC3b bound only to CRIgϩ naRPMs (Fig. 2A). C3 did not panel). This indicates that CRIg was able to bind to complement- bind to either CRIgϩ or CRIg៮ macrophages in line with CRIg’s opsonized particles in the absence of integrin activation. Thus, selective recognition of a neoepitope exposed on cleaved, but not increased binding activity of CRIgϩ naRPMs to EACs is mediated native, C3 (21). CRIg blocking Ab or a soluble CRIg protein sig- through both CRIg and CR3 following incubation at 37°C. Finally, nificantly reduced iC3b binding to RPMs while CR3 blocking Ab in contrast to CRIg, CR3 is dependent on divalent cations to ef- or CRIg isotype control Ab had no effect on binding activity (Fig. ficiently bind to opsonized targets, indicating different require- 2B), confirming the requirement of CRIg, and not CR3, for binding ments for CRIg and CR3 receptor function. to monomeric iC3b. For studying internalization of C3 proteins, freshly lavaged RPMs were cultured overnight to generate ad- CRIg localizes to phagosomes and enhances phagocytosis in RPMs. When incubated with cultured adRPMs at 37°C, the bound adRPMs ligands C3b and iC3b were internalized and colocalized with To further elucidate the subcellular localization and contribution of CRIgϩ vesicles in the areas surrounding the nucleus as well as at CRIg and CR3 to particle phagocytosis, adRPMs, capable of the tips of macrophage pseudopodia (inset, Fig. 2C). Residual up- phagocytosing complement-opsonized particles (27), were used. 7906 CRIg AND CR3 IN COMPLEMENT-MEDIATED PHAGOCYTOSIS

FIGURE 5. CRIg enhances phagocytosis in adRPMs. A, adRPMs, cultured overnight, were incubated with A488-conjugated EACs for the indicated time points at 37°C. adRPMs were then dissociated from the tissue culture plates and stained with a non-blocking Ab to CRIg. EACs bound to the cell surface were lysed and the extracellular green fluorescence was quenched using polyclonal anti-A488 Abs. The amount of phagocytosed EACs was then determined by the MFI of A488 using flow cytometry. B, adRPMs were incubated with A488-conjugated EACs for 30 min in the presence of isotype or CRIg and/or CR3 blocking Abs. The amount of phagocytosed EACs in CRIgϩ and CRIgϪ adRPMs was then determined by staining the cells with a non-blocking Ab to CRIg and measuring the p Ͻ 0.01. C, adRPMs were incubated with EACs (green) for ,ءء ;p Ͻ 0.05 ,ء .MFI of A488 using flow cytometry. Results are expressed as mean Ϯ SD, n ϭ 3 30 min in the presence of isotype (ϩ Control Ab) or CRIg blocking Abs (ϩ CRIg Ab) followed by staining with a non-blocking Ab to CRIg (red). Blue indicates nuclear staining with DAPI. In the presence of control Ab, CRIg accumulates at sites where the EACs contact the macrophage plasma membrane (arrows). In the presence of CRIg blocking Abs (ϩ CRIg Ab), CRIg accumulation at the sites of EAC contact is prevented. Scale bar ϭ 10 ␮m.

CR3 was distributed equally on all adRPMs while CRIg, as ex- Abs and then stained with a nonblocking Ab to CRIg. In the pres- pected, localized to a subset of adRPMs (Fig. 4A). To participate ence of isotype control Ab, CRIg accumulated in the forming in all steps of the phagocytic process, the receptors involved have phagosome (Fig. 5C, upper panels). Addition of CRIg-blocking to redistribute to the site of particle contact (30). Upon incubation Ab prevented CRIg accumulation in the phagosome and resulted in with EACs, CRIg and CR3 redistributed to localize in the phago- even distribution of CRIg on the cell surface (Fig. 5C, lower pan- some surrounding the EAC (Fig. 4B). To further determine els). This indicates that blocking CRIg binding to its ligand iC3b whether CRIg and CR3 colocalization with phagosomes translates prevents CRIg accumulation in the phagosome and inhibits phago- into more efficient phagocytosis, adRPMs were incubated with cytosis of the complement-opsonized particles. A488-fluorescently labeled EACs (Fig. 5A) followed by dissocia- In vivo, tissue macrophages often encounter various activating tion of the phagocytes from the culture dish. Phagocytosis was stimuli, including bacterial cell wall products and cytokines (31). determined by FACS analysis after lysis of cell surface-bound To determine whether CRIg could act as a phagocytic receptor EACs. Remaining fluorescence resulting from lysed EAC bound to once the macrophages were activated, adRPMs were incubated the cell surface was quenched with a polyclonal anti-A488 Ab. As overnight with LPS. This stimulation resulted in increased expres- a result, internalized EACs were the primary source of fluores- sion of the activation marker CD86, while CRIg expression was cence. Uptake of EACs was significantly increased in CRIgϩ com- slightly reduced (Fig. 6A). In the activated macrophages, particle pared with CRIgϪ adRPMs (Fig. 5A). In CRIgϩ adRPMs incu- internalization was evident only after 20 min (Fig. 6B). Similar to bated with EACs for 30 min, CRIg and CR3 each contributed to nonactivated macrophages, LPS-activated CRIgϩ adRPMs ϳ50% of total phagocytosis which was reduced to background showed significantly increased phagocytosis at 30 min, contributed levels when both receptors were simultaneously blocked (Fig. 5B, to by both CRIg and CR3 (Fig. 6C). Thus, CRIg significantly left panel). In CRIgϪ adRPMs, as expected, phagocytosis was en- contributes to increased complement-mediated phagocytosis in tirely mediated through CR3 (Fig. 5B, right panel). These results both nonactivated and LPS-activated adRPMs. indicate that CRIg and CR3 act independently and additively to enhance binding and phagocytosis of complement C3-opsonized Discussion particles. To determine whether CRIg blocking Ab disrupts traf- CRIg was initially described as a Kupffer cell-expressed CR re- ficking of CRIg to the forming phagosome, cultured adRPMs in- quired for clearance of pathogens from the circulation (20). This cubated with EACs were treated with isotype or CRIg-blocking study shows that, besides Kupffer cells, CRIg is expressed on a The Journal of Immunology 7907

FIGURE 6. CRIg is expressed and functional on LPS-activated adRPMs. A, adRPMs cultured overnight in the presence of 1 ␮g/ml LPS were incu- bated with A488-conjugated EACs as described in the legend of Fig. 5. FACS analysis was performed using an Ab to CD86, CD11b, and CRIg. B, Phagocy- tosis of A488-labeled EACs by LPS- activated adRPMs was determined at various time points as described in the legend of Fig. 5. C, LPS-activated ad- RPMs were incubated with A488-con- jugated EACs for 30 min in the pres- ence of isotype or CRIg and/or CR3 blocking Abs. The amount of phagocy- tosed EACs in CRIgϩ and CRIgϪ ad- RPMs was then determined by staining the cells with a non-blocking Ab to CRIg and measuring the MFI of A488 using flow cytometry. Results are ex- p Ͻ ,ء .pressed as mean Ϯ SD, n ϭ 3 .p Ͻ 0.01 ,ءء ;0.05 subpopulation of RPMs. These mononuclear phagocytes are capa- for the difference in these previous reports as compared with our ble of local synthesis of complement components (2, 32), enabling current observations is that only a small portion (Ͻ20%) of EACs opsonization of target particles in the absence of direct exposure to bound to the macrophage surface was internalized. Alternatively, serum. In the current study, we demonstrate that the two CRs ex- in our studies adRPMs were cultured overnight to assure firm ad- pressed on these macrophages, CRIg and CR3, independently con- hesion, a procedure that may have induced activation of the ad- tribute to complement-mediated phagocytosis of iC3b-opsonized RPMs sufficient to enable phagocytosis. Finally, in support of our particles. We further illustrate that the requirements for binding observations, adRPMs can internalize constituents of C3b-coated and ingestion of target particles differ between CRIgϩ and sheep erythrocyte membranes (38), indicating that non-stimulated CRIgϪ RPMs. adRPMs are capable of phagocytosis of complement C3-opsonized CRIg accelerates complement-mediated phagocytosis by rapidly particles. binding and internalizing opsonized particles independent of re- Different from other CRs, CRIg is present on recycling endo- ceptor crosslinking, activating stimuli or divalent cations. Thus, somes in adRPMs as well as in -derived human macro- ␣ ␤ CRIg functions differently from CR3/ M 2 integrin, which firmly phages (20). Endosomes are important in trafficking of plasma binds and internalizes opsonized targets only after exposing to ad- membrane and for cytoskeleton assembly required for phagosome ditional stimuli such as phorbol esters, chemokines, and TNF-␣ formation (39, 40). Their recruitment to the site of particle contact that induce major conformational changes in both the ␣- and may mediate incorporation of CRIg in the forming phagosome ␤-chains (10, 33). Although CRIg colocalizes with CR3 on a sub- ensuring close contact of the macrophage plasma membrane with population of RPMs, CRIg is absent from inflammatory macro- the opsonized particle. Indeed, Abs that blocked CRIg binding to phages infiltrating the peritoneum following a challenge with the iC3b prevented recruitment of CRIg to the phagosomes, resulting irritant thioglycollate (24) (results not shown). In the infiltrating in reduced phagocytosis of the opsonized target particles (Fig. 5). cells, CR3 is the dominant phagocytic receptor for C3 fragments Whether the recruitment of CRIgϩ endosomes to the forming (9, 15, 34). This indicates that CRIg functions to enhance phago- phagosome is an active process is unknown. CRIg has a relative cytosis only in resident, non-inflammatory tissue macrophages, large cytoplasmic tail containing several tyrosine residues that can whereas CR3 functions as the dominant CR in activated macro- potentially serve as docking sites for signaling components or cy- phages and neutrophils recruited to the sites of inflammation. CRIg toskeletal elements. The participation of CRIg in signaling path- expression therefore associates with a subpopulation of resident ways linking cytoskeletal reorganization to phagocytosis is an area macrophages that likely participate in phagocytosis of C3-opso- of current investigation. nized particles before phlogistic events that shape the ultimate im- Since in vivo tissue resident macrophages are continuously ex- mune response. The capability of CRIg to increase phagocytosis in posed to activating stimuli, including bacterial components from adRPMs and its absence from inflammatory macrophages fits well enteric microflora (41), we determined whether CRIg expression with a function in immune clearance and tissue homeostasis (35). or phagocytic function changes in vitro following activation of Furthermore, CRIg has been described on synovial lining macro- adRPMs with LPS. Although cell surface expression of CRIg was phages of arthritic joints but was absent from infiltrating sublining slightly reduced under these circumstances, CRIg remained func- macrophages (36). This fits with our observation that while CRIg tional as a phagocytic receptor as shown by a similar contribution expression is not lost upon LPS-stimulation of resident peritoneal of CRIg to EAC phagocytosis in nonstimulated as well as stimu- macrophages (Fig. 6A)orL. monocytogenes stimulation of Kupffer lated adRPMs. Thus, while CRIg is absent from inflammatory cells (20), it is absent on newly recruited inflammatory macrophages, it remains expressed and functional when tissue res- macrophages. ident macrophages are exposed to activating stimuli. The ability of adRPMs to phagocytose EACs is at variance with In a recent study (42), it was demonstrated that CRIg clears previous reports (29, 37) in which CR for C3b/iC3b on adRPMs complement-opsonized lacking DAF and Crry, for the has been shown to only bind and not internalize EACs. A reason first time illustrating a role of CRIg in clearance of self cells. 7908 CRIg AND CR3 IN COMPLEMENT-MEDIATED PHAGOCYTOSIS

Unexpectedly, complement C3-coated erythrocytes were not 17. Gordon, S., and P. R. Taylor. 2005. Monocyte and macrophage heterogeneity. cleared through a CRIg-dependent mechanism. A likely explana- Nat. Rev. Immunol. 5: 953–964. 18. Mevorach, D., J. O. Mascarenhas, D. Gershov, and K. B. Elkon. 1998. Comple- tion for this finding is that complement opsonization of the DAF ment-dependent clearance of apoptotic cells by human macrophages. J. Exp. and Crry double-deficient erythrocytes was much less efficient Med. 188: 2313–2320. 19. Gregory, S. H., L. P. Cousens, N. van Rooijen, E. A. Dopp, T. M. Carlos, and compared with opsonization, resulting in a significantly E. J. Wing. 2002. Complementary adhesion molecules promote - reduced interaction with CRIg. In the present study, IgM anti- Kupffer cell interaction and the elimination of bacteria taken up by the . J. Forsmann Ag was used to provide optimal complement activation Immunol. 168: 308–315. 20. Helmy, K. Y., K. J. Katschke, Jr., N. N. Gorgani, N. M. Kljavin, J. M. Elliott, on the erythrocyte surface leading to high levels of C3 deposition L. Diehl, S. J. Scales, N. Ghilardi, and M. van Lookeren Campagne. 2006. CRIg: (Fig. 3A), comparable to what was found on the platelets (42). a macrophage complement receptor required for phagocytosis of circulating Thus, next to a potential influence of the nature of the particle, the pathogens. Cell 124: 915–927. 21. Wiesmann, C., K. J. Katschke, J. Yin, K. Y. Helmy, M. Steffek, W. J. Fairbrother, degree of C3 opsonization is an important determinant for clear- S. A. McCallum, L. Embuscado, L. DeForge, P. E. Hass, and ance by CRIg. M. van Lookeren Campagne. 2006. Structure of C3b in complex with CRIg gives insights into regulation of complement activation. Nature 444: 217–220. Together, CRIg identifies a novel subpopulation of macrophages 22. Pepys, M. B., A. C. Dash, A. H. Fielder, and D. D. Mirjah. 1977. Isolation and with a high capacity for complement-mediated phagocytosis in the study of murine C3. Immunology 33: 491–499. absence and presence of activating stimuli. These studies illustrate 23. Circolo, A., G. Garnier, W. Fukuda, X. Wang, T. Hidvegi, A. J. Szalai, D. E. Briles, J. E. Volanakis, R. A. Wetsel, and H. R. Colten. 1999. Genetic the functional diversity of tissue resident macrophages and will disruption of the murine complement C3 region generates deficient further guide research to understand the role of CRIg in tissue mice with extrahepatic expression of C3 mRNA. Immunopharmacology 42: homeostasis and host defense. 135–149. 24. Vogt, L., N. Schmitz, M. O. Kurrer, M. Bauer, H. I. Hinton, S. Behnke, D. Gatto, P. Sebbel, R. R. Beerli, I. Sonderegger, et al. 2006. VSIG4, a B7 family-related Acknowledgments protein, is a negative regulator of activation. J. Clin. Invest. 116: 2817–2826. We thank Drs. Rick Brown, Wouter Hazenbos, Nico Ghilardi, and Paul 25. Molina, H., W. Wong, T. Kinoshita, C. Brenner, S. Foley, and V. M. Holers. Godowski for insightful discussions. We are grateful to Jim Cupp, Michael 1992. Distinct receptor and regulatory properties of recombinant mouse comple- Hamilton, Susan Palmieri, Laszlo Komuves, and Meredith Sagolla for their ment receptor 1 (CR1) and Crry, the two genetic homologues of human CR1. J. Exp. Med. 175: 121–129. contributions. 26. Martin, B. K., and J. H. Weis. 1993. Murine macrophages lack expression of the Cr2–145 (CR2) and Cr2–190 (CR1) gene products. Eur. J. Immunol. 23: Disclosures 3037–3042. 27. Brown, E. J. 1991. Complement receptors and phagocytosis. Curr. Opin. Immu- All authors are current or former employees of Genentech. nol. 3: 76–82. 28. Arnaout, M. A., S. L. Goodman, and J. P. Xiong. 2007. Structure and mechanics of integrin-based cell adhesion. Curr. Opin. Cell Biol. 19: 495–507. References 29. Bianco, C., F. M. Griffin, Jr., and S. C. Silverstein. 1975. Studies of the macro- 1. Stuart, L. M., and R. A. Ezekowitz. 2005. Phagocytosis: elegant complexity. phage complement receptor: alteration of receptor function upon macrophage Immunity 22: 539–550. activation. J. Exp. Med. 141: 1278–1290. 2. Carroll, M. C. 2000. The role of complement in activation and tolerance. 30. Griffin, F. M., Jr., J. A. Griffin, J. E. Leider, and S. C. Silverstein. 1975. Studies Adv. Immunol. 74: 61–88. on the mechanism of phagocytosis. I. Requirements for circumferential attach- 3. Walport, M. J. 2001. Complement: first of two parts. N. Engl. J. Med. 344: ment of particle-bound ligands to specific receptors on the macrophage plasma 1058–1066. membrane. J. Exp. Med. 142: 1263–1282. 4. Dempsey, P. W., M. E. Allison, S. Akkaraju, C. C. Goodnow, and D. T. Fearon. 31. Stout, R. D., and J. Suttles. 2004. Functional plasticity of macrophages: reversible 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired adaptation to changing microenvironments. J. Leukocyte Biol. 76: 509–513. immunity. Science 271: 348–350. 32. Newell, S. L., D. C. Shreffler, and J. P. Atkinson. 1982. Biosynthesis of C4 by 5. Liszewski, M. K., T. C. Farries, D. M. Lublin, I. A. Rooney, and J. P. Atkinson. mouse peritoneal macrophages. I. Characterization of an in vitro culture system 1996. Control of the . Adv. Immunol. 61: 201–283. and comparison of C4 synthesis by “low” vs “high” C4 strains. J. Immunol. 129: 6. Cabanas, C., and F. Sanchez-Madrid. 1999. CD11c (leukocyte integrin CR4 ␣ 653–659. subunit). J. Biol. Regul. Homeostatic Agents 13: 134–136. 33. Xiong, J. P., T. Stehle, S. L. Goodman, and M. A. Arnaout. 2003. New insights 7. Ehlers, M. R. 2000. CR3: a general purpose adhesion-recognition receptor es- into the structural basis of integrin activation. Blood 102: 1155–1159. sential for innate immunity. Microbes Infect. 2: 289–294. 34. Drevets, D. A., and P. A. Campbell. 1991. Roles of complement and complement 8. Gasque, P. 2004. Complement: a unique innate immune sensor for danger signals. receptor type 3 in phagocytosis of Listeria monocytogenes by inflammatory Mol. Immunol. 41: 1089–1098. mouse peritoneal macrophages. Infect. Immun. 59: 2645–2652. 9. Ross, G. D., and V. Vetvicka. 1993. CR3 (CD11b, CD18): a phagocyte and NK 35. He, J. Q., C. Wiesmann, and M. van Lookeren Campagne. 2008. A role of mac- cell membrane receptor with multiple ligand specificities and functions. Clin. rophage complement receptor CRIg in immune clearance and inflammation. Mol. Exp. Immunol. 92: 181–184. Immunol. 45: 4041–4047 10. Luo, B. H., C. V. Carman, and T. A. Springer. 2007. Structural basis of integrin 36. Lee, M. Y., W. J. Kim, Y. J. Kang, Y. M. Jung, Y. M. Kang, K. Suk, J. E. Park, regulation and signaling. Annu. Rev. Immunol. 25: 619–647. E. M. Choi, B. K. Choi, B. S. Kwon, and W. H. Lee. 2006. Z39Ig is expressed 11. Wright, S. D., and B. C. Meyer. 1986. Phorbol esters cause sequential activation on macrophages and may mediate inflammatory reactions in arthritis and athero- and deactivation of complement receptors on polymorphonuclear leukocytes. sclerosis. J. Leukocyte Biol. 80: 922–928. J. Immunol. 136: 1759–1764. 37. Griffin, F. M., Jr., C. Bianco, and S. C. Silverstein. 1975. Characterization of the 12. Vetvicka, V., B. P. Thornton, and G. D. Ross. 1996. Soluble ␤-glucan polysac- macrophage receptor for complement and demonstration of its functional inde- charide binding to the lectin site of neutrophil or natural killer cell complement pendence from the receptor for the Fc portion of immunoglobulin G. J. Exp. Med. receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable 141: 1269–1277. of mediating cytotoxicity of iC3b-opsonized target cells. J. Clin. Invest. 98: 38. de Water, R., L. A. Ginsel, W. T. Daems, and M. R. Daha. 1983. Autoradio- 50–61. graphical demonstration of C3b receptor activity on resident peritoneal macro- 13. Li, B., D. J. Allendorf, R. Hansen, J. Marroquin, C. Ding, D. E. Cramer, and phages. Histochemistry 77: 289–298. J. Yan. 2006. Yeast ␤-glucan amplifies phagocyte killing of iC3b-opsonized tu- 39. Bajno, L., X. R. Peng, A. D. Schreiber, H. P. Moore, W. S. Trimble, and mor cells via complement receptor 3-Syk-phosphatidylinositol 3-kinase pathway. S. Grinstein. 2000. Focal exocytosis of VAMP3-containing vesicles at sites of J. Immunol. 177: 1661–1669. phagosome formation. J. Cell Biol. 149: 697–706. 14. Drevets, D. A., P. J. Leenen, and P. A. Campbell. 1993. Complement receptor 40. Cox, D., D. J. Lee, B. M. Dale, J. Calafat, and S. Greenberg. 2000. A Rab11- type 3 (CD11b/CD18) involvement is essential for killing of Listeria monocyto- containing rapidly recycling compartment in macrophages that promotes phago- by mouse macrophages. J. Immunol. 151: 5431–5439. cytosis. Proc. Natl. Acad. Sci. USA 97: 680–685. 15. Rothlein, R., and T. A. Springer. 1985. Complement receptor type three-depen- 41. Bilzer, M., F. Roggel, and A. L. Gerbes. 2006. Role of Kupffer cells in host dent degradation of opsonized erythrocytes by mouse macrophages. J. Immunol. defense and liver disease. Liver Int. 26: 1175–1186. 135: 2668–2672. 42. Kim, D. D., T. Miwa, Y. Kimura, R. A. Schwendener, M. van Lookeren 16. Cailhier, J. F., M. Partolina, S. Vuthoori, S. Wu, K. Ko, S. Watson, J. Savill, Campagne, and W. C. Song. 2008. Deficiency of decay-accelerating factor and J. Hughes, and R. A. Lang. 2005. Conditional macrophage ablation demonstrates -related gene/protein y on murine platelets leads to com- that resident macrophages initiate acute peritoneal inflammation. J. Immunol. plement-dependent clearance by the macrophage phagocytic receptor CRIg. 174: 2336–2342. Blood 112: 1109–1119.